Antimicrobial polyanhydride nanoparticles

ABSTRACT

The invention provides compositions and methods to treat microbial infections in animals, to inhibit the replication of microbes in infected cells, and to kill pathogens in infected cells. The methods can include administering to an animal in need of such treatment an effective antimicrobial amount of a composition comprising polyanhydride microparticles or nanoparticles that encapsulate a plurality of antimicrobial agents. The polyanhydride microparticles or nanoparticles can be, for example, copolymers of sebacic anhydride (SA) and 1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride, copolymers of 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) anhydrides and 1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride, or various combinations thereof. The microparticles or nanoparticles can accumulate in infected monocytes, dendritic cells, both, or on or in other infected cells, and degrade by surface erosion over a period of time to release the antimicrobial agents, thereby killing or inhibiting the microbes and treating the infection.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/940,872, filed Nov. 5, 2010, and this application claims priority toU.S. Provisional Patent Application No. 61/259,061, filed Nov. 6, 2009,the specifications of which are incorporated herein by reference.

BACKGROUND

Chronic bacterial infections remain a significant cause of morbidity andmortality in human and animal populations. Current research effortsfocus on generating effective preventative measures through vaccinedevelopment and on developing new antimicrobial agents to overcomerapidly increasing numbers of resistant microbes. Despite these efforts,the overall incidence of antibiotic resistance associated with chronicbacterial diseases continues to rise.

Intracellular pathogens, such as Mycobacterium, Salmonella, Chlamydia,Borrelia, Rickettsia, and Brucella, are particularly difficult to treatbecause they infect an intracellular niche inside of cells that protectsthem from being exposed to extracellular host defenses and highconcentrations of antibiotics.

A specific example of a difficult to treat intracellular microbialpathogen is Brucella spp. Brucella are facultative intracellularpathogens of humans and domestic animals. Human brucellosis is strictlyzoonotic and manifests as a chronic, debilitating disease from whichthere is no protective immunity. Brucellosis in humans is considered themost common zoonotic infection worldwide. Human brucellosis has emergedin new areas of the world, particularly in central Asia, while numbersof cases in endemic areas have not been reduced.

Despite the successful use of live-attenuated strains for vaccination inanimals, no human vaccine is available for human brucellosis, andvaccine strains approved for use in animals are pathogenic to humans.These disease characteristics contribute to the bacterium being listedas a Category B Bioterrorism Agent. A key to Brucella pathogenesis isthe organism's ability to survive and replicate within host monocytesand macrophages. Virulent Brucella prevents the fusion of phagosomeswith lysosomes. The bacteria then replicate in a secondary intracellularcompartment that is not acidic and that is removed from normal vesicletrafficking pathways.

Antibiotic resistance is not a hallmark of persistence within the hostfor intracellular pathogens compared to extracellular pathogens. Aremarkably small number of bacteria have adapted to survive andreplicate within monocytes and macrophages by modifying the partitioningof the subcellular compartments within the infected cell to avoiddegradation and go on to replicate. The adaptation to the intracellularenvironment provides protection by sequestration from most immunedefenses. Species of several genera have adapted in such a manner,including Mycobacterium, Yersinia, Francisella, Brucella, Burkholderia,Salmonella, Bordetella, and Erhlichia. During the chronic stage ofBrucellosis, the bacteria persist and replicate in tissue residentmacrophages. Reaching bactericidal concentrations in intracellularenvironments has proven very difficult using currently used methods.Standard antibiotic regimens for treatment of Brucellosis often requiretwo to three antibiotics given simultaneously for a minimum of 6 weeks.These regimen are difficult to follow and are often ineffective, andpatient compliance is a significant problem.

Accordingly, new antimicrobial formulations are needed, such asformulations that can target and/or deliver antimicrobial agents to theintracellular environment of cells infected with bacterial pathogens.Antimicrobial formulations that provide enhanced bactericidal activityare also eagerly sought. Compositions and methods that renderantibiotics effective during the chronic stages of a disease are alsoneeded in the art.

SUMMARY

Polyanhydride particles (PA particles) such as microspheres andnanospheres can elicit unique cellular responses from immune cells. ThePA particles stimulate internalization, direct intracellular traffickingand degrade slowly within the cells. Antimicrobial compounds can beencapsulated into PA particles, thereby allowing for the compounds to beslowly released after they are internalized by cells as the particleslowly degrades by surface erosion. Varying the type of copolymerconstituents of the particle effects particle degradation and can alterthe fate of the particle within cells.

Microbial pathogens survive within host tissues by protecting themselvesagainst immune defenses. For example, intracellular pathogens evade hostdefenses by adapting themselves to the environment within cells, whichallows the pathogens to escape contact with antibiotics. PA particlescan enter host cells and deliver antibiotics to the samemicroenvironment of the pathogen. The highly effective targeting of theintracellular environment by PA particles greatly reduces the amount ofantibiotic needed to treat such an infection. PA particles also providedelayed and slow release of the encapsulated drug. As described herein,PA particles (nanospheres) that have encapsulated doxycycline witheither 1.5% or 3% loading effectively kill intracellular pathogens,including laboratory and field strains of Brucella canis and laboratorystrains of Escherichia coli, as determined by agar disk diffusionassays.

Accordingly, the invention provides a polyanhydride microparticle ornanoparticle that contains a plurality of antimicrobial agents insidethe particle; wherein the polyanhydride nanoparticle comprises anhydridecopolymers of a 1,ω-bis(carboxy)(C₂-C₁₀)alkane and a1,ω-bis(carboxyphenoxy)(C₂-C₁₀)alkane. The nanoparticle can besubstantially spherical in shape and can have an average diameter ofabout 100 nm to about 900 nm. When the particle is a microparticle, themicroparticle can be substantially spherical in shape and can have anaverage diameter of about 900 nm to about 5 μm.

The 1,ω-bis(carboxy)(C₂-C₁₀)alkane can be sebacic anhydride (SA),1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) anhydride, or acombination thereof. The 1,ω-bis(carboxyphenoxy)(C₂-C₁₀)alkane can be,for example, 1,6-bis-(p-carboxy-phenoxy)hexane (CPH). The polyanhydridenanoparticle is formed from anhydrides of these components for formcopolymers. The ratio of 1,ω-bis(carboxy)(C₂-C₁₀)alkane to1,ω-bis(4-carboxyphenoxy)(C₂-C₁₀)alkane in the nanoparticle can be about90:10 to about 50:50 to about 10:90, or any ratio in between, such as85:15, 80:20, 75:25, 70:30, 60:40, or 55:45, or the reverse of suchratios.

In certain specific embodiments, the 1,ω-bis(carboxy)(C₂-C₁₀)alkane issebacic anhydride (SA) and the 1,ω-bis(carboxyphenoxy)(C₂-C₁₀)alkane is1,6-bis-(p-carboxyphenoxy)hexane (CPH). The1,ω-bis(carboxy)(C₂-C₁₀)alkane can also be1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and the1,ω-bis(carboxyphenoxy)(C₂-C₁₀)alkane can be1,6-bis-(p-carboxyphenoxy)hexane (CPH). The antimicrobial agent can bedoxycycline or another antimicrobial agent, as described below. Thenanoparticle can further include a second antimicrobial agent, asdescribed below.

The invention also provides a method to treat a microbial infection inan animal. The method can include administering to an animal in need ofsuch treatment an effective antimicrobial amount of a composition thatincludes polyanhydride microparticles or nanoparticles that contain oneor more antimicrobial agents. The composition can include theantimicrobial polyanhydride particles and, for example, apharmaceutically acceptable excipient, diluent or carrier.

The polyanhydride microparticles or nanoparticles can comprisecopolymers of, for example, sebacic anhydride (SA) and1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride. The microparticles ornanoparticles then accumulate in infected monocytes, dendritic cells, orboth, and degrade by surface erosion over a period of time to releasethe antimicrobial agents; so as to treat the microbial infection.

The microbial infection can be an infection that causes a chronicdisease. For example, the infection can be a bacterial infection. Thebacterial infection can be caused by, for example, bacteria of thegenera Bordetella, Borrelia, Brucella, Burkholderia, Chlamydia,Erhlichia, Francisella, Mycobacterium, Rickettsia, Salmonella, and/orYersinia. The microbial infection may be one that causes Bacterialmeningitis, Brucellosis, Erhlichiosis, Glanders, Johne's, mastitis,Legionella, Lyme disease, Mycobacteria disease complex, Mycoplasmosis,Q-fever, Salmonellosis, Shigellosis, or Tuberculosis.

In one embodiment, the antimicrobial agent can be doxycycline,optionally in the presence of a second agent, such as bacillomycin.Other antimicrobial agents are further described below. In someembodiments, the polyanhydride microparticles or nanoparticles canencapsulate an average of about 1 μg to about 12 μg of the antimicrobialagent per mL or per particle. The polyanhydride microparticles ornanoparticles further comprise an additional antimicrobial agent. Theadditional antimicrobial agent can be any other suitable agent, forexample, a different type of antimicrobial agent, a therapeutic smallmolecule (e.g., an antimicrobial agent of less than 2 kDa) or a heavymetal. Examples of heavy metal therapeutic agents include copper, iron,aluminum, zinc, gold, compound and ions thereof, and variouscombinations thereof.

The invention also provides a method to deliver antimicrobial agents tocells infected with microbes comprising contacting cells infected bymicrobes with an effective amount of a composition comprisingpolyanhydride microparticles or nanoparticles that encapsulate aplurality of antimicrobial agents;

wherein the polyanhydride microparticles or nanoparticles comprisecopolymers of sebacic anhydride (SA) and1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride; and

-   -   wherein the microparticles or nanoparticles accumulate in the        cells infected by microbes, and degrade by surface erosion over        a period of time to release the antimicrobial agents; thereby        inhibiting the replication of the microbes, killing the        microbes, or both.

The invention further provides improved delivery of antimicrobialagents, such as antibiotics, into host cells that contain pathogens,compared to known delivery systems. Prior attempts to use packagedantibiotics have failed to kill pathogens more effectively than freeantibiotics alone (e.g., treatment by standard therapy).

The invention also provides greatly enhanced uptake of PA particlescompared to PLGA micro- or nanospheres. Base catalyzed degradationincreases the particle longevity within tissues and cells, leading tolonger release profiles of bioavailable antimicrobial agents. The PAparticles also provide greater encapsulation of hydrophobic antibioticscompared to hydrophilic PLGA-based particles. The acidic compartmentprovided by hydrophilic PLGA-based particles is detrimental to mostantibiotics. However, the PA particle interior is significantly lessacidic, thereby providing an encapsulated environment for antimicrobialagents that is much less acidic than PLGA-based particles, which istherefore less detrimental to many kinds of antimicrobial agents thanPLGA-based particles.

The chemistry of polyanhydride particles modulate cellular responsesthat specifically effect cellular uptake, vesicular trafficking anddelivery to intracellular organelles. These factors contribute to thetype of microenvironment that forms around internalized particles aswell as the subsequent release of the antibiotic resulting from thebreakdown of the polymerized subunits within the particles themselves.Thus, the chemistry of particles (e.g., the hydrophobicity of theparticle interior) serves as an excellent delivery vehicle environmentfor antibiotics because the chemical properties modulate uptake and aidthe determination of the particles' fate within the cell. Theseparticles can be embedded with current antibiotics already approved astreatments for numerous diseases. Loading of antimicrobial agents intoPA particles does not chemically modify the antibiotics or negate theirantimicrobial function.

For example, cargo loaded PA particles can penetrate and localize to theintracellular compartment occupied by Mycobacterium paratuberculosisusing tissue culture cells previously infected with the bacteria. Suchlocalization significantly increases the effectiveness of the cargo,thereby reducing the amount of antimicrobial agent necessary to treatthe infection.

Experimental observations reveal that the polyanhydride particlesdistribute inside cells in a similar pattern to that observed forseveral intracellular pathogens. Thus, the intracellular trafficking ofpathogens allowed for the recognition that polyanhydride particles canbe used to deliver antimicrobial agents to a pathogen's previously safeintracellular compartment to eliminate the pathogen from the hostentirely. The PA particles described herein can therefore be used totreat human diseases, such as Tuberculosis, Brucellosis, Lyme disease,Shigellosis, Q-fever, Glanders, Legionella, Erhlichiosis, Salmonellosis,and Bacterial meningitis, as well as the corresponding infections inother animals, including but not limited to Brucellosis, Mycoplasmosis,Johne's, mastitis, Mycobacteria complex, and Bacterial meningitis.

The invention therefore provides antimicrobial agents encapsulated innanospheres. The nanospheres can enter the intracellular environment ofinfected cells. The formulations described herein provide for improvedintracellular targeting and release of encapsulated antimicrobial agentswithin infected cells, such as Brucella infected cells, leading toenhanced antimicrobial activity. This increase in antimicrobial activitycan make antibiotics significantly more effective during the chronicstages of a disease when most patients begin showing clinical symptoms,leading them to seek medical attention. Once the chronic stage isreached, use of a single antibiotic to treat the disease most oftenfails to eliminate pathogen from the host. Antimicrobial agents can beeffectively targeted to specific intracellular niches of variousintracellular pathogens using the polyanhydride nanospheres describedherein.

The invention provides for the use of the compositions described hereinfor use in medical therapy. The medical therapy can be treatinginfections, for example, bacterial, fungi, algal, or other pathogenicinfections. Examples of such infections include Tuberculosis,Brucellosis, Lyme disease, Shigellosis, Q-fever, Glanders, Legionella,Erhlichiosis, Salmonellosis, bacterial sepsis and bacterial meningitis.The invention also provides for the use of a composition as describedherein for the manufacture of a medicament to treat microbial infectionsin animals, for example, mammals, such as humans. The medicament caninclude a pharmaceutically acceptable diluent, excipient, or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention, however, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1 illustrates two hour counts of particles internalized/dendriticcell in dendritic cells and monocytes.

FIG. 2 illustrates 48 hour counts of particles internalized/dendriticcell in dendritic cells and monocytes.

FIG. 3 illustrates intracellular compartment localized to lysosomes byLAMP1 staining.

FIG. 4 illustrates intracellular localization of Brucella abortus withinmonocytes (same lineage as dendritic cells).

FIG. 5 illustrates quantifying the intracellular stability ofnanospheres using morphometric image analysis. Images ofFITC-encapsulated nanospheres were captured using 40× objective andprocessed using constant values for camera exposure and imagethresholding throughout the experiment. Images of Lamp1 and nucleistained dendritic cells incubated with nanospheres at indicated timeswere background subtracted to generate binary equivalent image (FITCBinary) to perform particle analysis using ImageJ v1.42 software (bar=5μm).

FIG. 6 illustrates binary particle counts and pixel area results asdetermined from FIG. 1. Binary particle counts and pixel area resultswere averaged from at least 15 separate fields of view for each timepoint and particle composition.

FIG. 7 illustrates a data of chronic infection of Blk6 mice by virulentBrucella abortus strain 2308. The experiment illustrates theestablishment of infection at 1 week and how it is followed bymaintenance of the chronic infection in the spleens of mice infectedi.p. with Brucella. CFU of Brucella recovered from the spleens of theseanimals are shown as an average of 5 mice per group per time point.Student's T-test indicates that the KO mouse strain has fewer Brucella.

FIG. 8. Stimulation of monocytes with interferon-γ inhibits theintracellular replication of Brucella. Adherent THP-1 cells were treatedwith 200 U/mL of IFN-γ beginning 24 hours prior to infection withopsonized B. abortus. Percent survival of intracellular bacteria wasdetermined as described and representative results are shown.

FIG. 9 illustrates the enhanced uptake of soluble Eα-RFP antigen bymonocytes (nuclei lighter shade) after co-incubation with polyanhydridenanospheres for 2 hours. Data demonstrated that the poly(SA) nanospheresenhanced antigen internalization more readily than did 20:80 CPH:SAfollowed by 50:50 CPH:SA. Representative epifluorescent images werecaptured and processed using identical exposure and ImageJ softwaresettings. Adjacent bar graphs summarize the average amount of RFPdetected per cell. Pixel areas within each image correspond to relativeintensity of RFP signal detected inside cells. Values from threerandomly selected fields of view were used to calculate averages andstandard deviation. Scale bar=5 μm.

FIG. 10 illustrates scanning electron photomicrographs of a. poly(SA)nanospheres; b. 20:80 CPH:SA nanospheres; and c. 50:50 CPH:SAnanospheres. Scale bars=2 μm.

FIG. 11 illustrates confocal photomicrographs of FITC-labeledpolyanhydride nanospheres internalized by THP-1 cells. Adherentmonocytes were incubated with nanospheres for 30 minutes before cultureswere washed and continued to incubate for an additional 2 hours.Poly(SA) and 20:80 CPH:SA nanoparticles were internalized to a muchgreater extent than 50:50 CPH:SA nanospheres. The majority ofinternalized poly(SA) and 20:80 CPH:SA were bound by cholesterol richmembranes as indicated by the high degree of co-localization.Representative images were captured by LSCM. Lipid rafts were identifiedusing Alexa 555 CTx (Molecular Probes). Scale bar=5 μm.

FIG. 12 illustrates confocal images of the intracellular localization ofFITC-nanospheres in THP-1 cells 48 h after uptake. Representative imageswere captured by LSCM and processed using ImageJ. The majority ofinternalized poly(SA) and 20:80 CPH:SA nanospheres were bound bycholesterol rich membranes as indicated by the high degree ofco-localization. Acidic vesicles (two left columns) were identifiedusing the pH responsive Lysotracker dye and cholesterol rich lipid rafts(two right columns) were visualized using Alexa 555 conjugated CTx(Molecular Probes). Note the general absence of FITC 50:50 CPH:SAnanospheres compared to 20:80 CPH:SA and poly(SA). Scale bar=5 μm.

FIG. 13 illustrates the viability of intracellular B. abortus at 72hours post-inoculation after treatment with doxycycline solubilized byPBS solution (5% doxycycline in PBS) or encapsulated in variouspolyanhydride particles (equivalent mass of doxycycline as in PBSsolution).

FIG. 14 illustrates the continued antibiotic release by serial disctransfer of polyanhydride particles containing doxycycline, while PBSsolubilized doxycycline discs provided no zones of inhibition on daytwo.

FIG. 15 illustrates viability of Mycobacterium avium subsp.paratuberculosis (MAP) at 24 hours and 48 hours after treatment with PBSsolubilized amikacin or polyanhydride particles containing 10 μg/mL mgamikacin.

FIG. 16 illustrates viability of MAP in broth at 24 hours and 48 hoursafter treatment with PBS solubilized amikacin or polyanhydride particlescontaining 1.25 μg/mL amikacin.

FIG. 17 illustrates viability of MAP in broth at 24 hours and 48 hoursafter treatment with PBS solubilized amikacin or polyanhydride particlescontaining 10 μg/mL amikacin.

FIG. 18 illustrates a schematic showing how determinations ofintracellular bacterial viability and extracellular bacterial killingwere obtained in Example 9, according to various embodiments.

FIG. 19 illustrates percent viability of intracellular MAP in U937 humanpro-monocytes at 24 hours and 48 hours after treatment with PBSsolubilized amikacin or polyanhydride particles containing 10 μg/mLamikacin.

FIG. 20 illustrates percent viability of intracellular MAP in RAW264 at24 hours and 48 hours after treatment with PBS solubilized amikacin orpolyanhydride particles containing 10 μg/mL amikacin.

FIG. 21 illustrates percent viability of intracellular MAP in J774A at24 hours and 48 hours after treatment with PBS solubilized amikacin orpolyanhydride particles containing 10 μg/mL amikacin.

DETAILED DESCRIPTION

The intracellular environment is a privileged site, where the outermostmembrane of the host cell prevents large concentrations of solutes, suchas antimicrobial agents, present outside the cell from entering thecell. Even once the antimicrobial agents enters a cell, to be aneffective therapeutic treatment, the agent must preferably 1) accumulatein a large enough effective dose for, e.g., either bactericidal orbacteriostatic killing; 2) be resistant to inactivation by acidic pH orlysosomal enzymes; 3) deliver or accumulate within the samesubcompartment shared with the microbe; 4) lengthen the time ofantibiotic activity following a single dose; and 5) must not harm thehost.

Encapsulated antimicrobial agents delivered to an infected intracellularenvironment can improve the intracellular targeting, release, andeffectiveness of encapsulated antimicrobial agents within infectedcells, such as Brucella infected cells, leading to enhancedantimicrobial activity. For example, encapsulated antibacterial agentscan be used to delivers the agents to an infected intracellularenvironment, such as cells infected by Brucella, thereby providing anincrease in anti-Brucella activity. This increased activity can makeantibiotics effective during the chronic stages of the disease when mostpatients present with symptoms and current single antibiotic treatments.Antibiotics can be effectively targeted to the specific intracellularniche of various intracellular pathogens using polyanhydridenanospheres.

Antimicrobial agents encapsulated into polyanhydride (PA) nanospherescan be effective at treating animals persistently infected withmicrobes, such as Brucella. The combination of size and chemistry of thehydrophobic nanospheres synergize to produce a particle that is rapidlyinternalized and localized to the phagolysosomal compartment, followinga pattern of intracellular trafficking very similar to virulentBrucella. Within the phagolysosomal vesicle, PA nanospheres persist byresisting bulk degradation under the acidic conditions and slowlyrelease encapsulated antimicrobial agents into the intracellularenvironment. The PA encapsulation techniques can be used for theeffective delivery of a variety of antimicrobial agents (of first,second, third, fourth and subsequent derivative generations) for eachgroup, such as tetracycline, penicillin, sulfa, cephalosporin,aminoglycoside, macrolide, and fluoroquinolone antibiotics; for example,doxycycline, spectinomycin, gentamicin, vancomycin, ciprofloxacin,cephalexin and trimethoprim-sulfamethoxazole.

Intracellular pathogens can cause debilitating and often fatalinfections. The infections are frequently chronic and persistent.Infections cause by intracellular pathogens are difficult to treatbecause the pathogens reside in immune privileged sites within theinfected cells. For example, treating Brucella infected animals withsingle or combination antibiotics, even for extended periods of time,typically eliminates only symptoms associated with acute infections.Thus, current treatments for Brucella infected animals fall short ineliminating tissue resident bacteria. The treatment methods describedherein can eliminate microbes, such as bacteria, that persistintracellularly within tissue resident macrophages during the chronicstages of disease.

Brucella infections are difficult to diagnose due to several factorsrelated to the organism's pathogenesis. Such factors include difficultyin quantifying bacteria during a chronic stage, lack of accuratemeasures of successful treatment, and tissue localization. Brucellainfection is almost exclusively within tissues where detection ofbacteria is very difficult, given the low CFU and transient bacteremiadetected in peripheral blood, which is the preferred means forcollecting specimens from suspect cases. Additional difficulties includethe late onset of a disease (e.g., when patients present), and poordiagnostic tools to assess the bacterial burden. Practitioners often donot know when a treatment has been effective because it is quitedifficult to quantify the number of Brucella cells at any given time,without harvesting organs.

During the chronic stage of Brucellosis, the bacteria persist andreplicate in tissue resident macrophages. This intracellular niche hasproven extremely difficult to target effectively with antimicrobialtherapy. The delivery of encapsulated antibiotics to the intracellularenvironment improves intracellular targeting and release of encapsulatedantibiotics within Brucella infected cells, leading to enhancedbactericidal activity. Targeted intracellular delivery dramaticallyincreases anti-Brucella activity during the chronic phase of the diseasewhen most patients present with symptoms. Antibiotics can be effectivelytargeted to the specific intracellular niche of various intracellularpathogens using polyanhydride nanospheres.

Polyanhydride copolymer nanospheres (PANS) elicit cellular responsesfrom monocytes and dendritic cells that stimulate internalization anddirect intracellular trafficking. The PANS can carry cargo such asantimicrobial agents and can release the cargo by slow degradation ofthe particle within the cells as a form of controlled release. Varyingthe polymer chemistry of the particle effects particle degradation ratesand alters the fate of the particle within cells. PANS are capable ofentering host cells and delivering antibiotics in the samemicroenvironment of the pathogen. This highly effective targeting of theintracellular environment greatly reduces the amount of antibioticneeded to treat such an infection and also provides delayed release.

DEFINITIONS

As used herein, certain terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14^(th) Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,“an example embodiment”, etc., indicate that the embodiment describedmay include a particular aspect, feature, structure, moiety, orcharacteristic, but not every embodiment necessarily includes thataspect, feature, structure, moiety, or characteristic. Moreover, suchphrases may, but do not necessarily, refer to the same embodimentreferred to in other portions of the specification. Further, when aparticular aspect, feature, structure, moiety, or characteristic isdescribed in connection with an embodiment, it is within the knowledgeof one skilled in the art to affect such aspect, feature, structure,moiety, or characteristic in connection with other embodiments, whetheror not explicitly described.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only,” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer. Unless indicated otherwise herein, the term“about” is intended to include values, e.g., weight percents, proximateto the recited range that are equivalent in terms of the functionalityof the individual ingredient, the composition, or the embodiment.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percents or carbon groups) includes each specific value, integer,decimal, or identity within the range. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than,”“or more” and the like include the number recited and refer to rangeswhich can be subsequently broken down into subranges as discussed above.In the same manner, all ratios disclosed herein also include allsubratios falling within the broader ratio.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Additionally, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations andunderstood as being modified in all instances by the term “about.” Thesevalues can vary depending upon the desired properties sought to beobtained by those skilled in the art utilizing the present teachings ofthe present invention. It is also understood that such values inherentlycontain variability necessarily resulting from the standard deviationsfound in their respective testing measurements.

The phrase “one or more” is readily understood by one of skill in theart, particularly when read in context of its usage. For example, one ormore substituents on a phenyl ring refers to one to five, or one to upto four, for example if the phenyl ring is disubstituted.

The terms “polyanhydride particle” and “polyanhydride nanosphere” bothrefer to microparticles and nanoparticles made of polyanhydride polymersas described herein. The polyanhydride polymers of the particles aretypically copolymers, such as random mixes of anhydride oligomers(condense prepolymers). The polyanhydride particle can be abbreviated as“PA particle”, which can be a microparticle or a nanoparticle. Thenanoparticles can also be referred to as polyanhydride nanosphere(PANS).

The group “alkyl” refers to a linear or branched hydrocarbon radical ordiradical that is optionally unsaturated and optionally substituted withfunctional groups as described herein. The alkyl group can contain 1 toabout 20 carbon atoms. Typical alkyl groups include, but are not limitedto, methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, 3-pentyl, hexyl, heptyl, octyl, or decyl. In oneembodiment, alkyl is preferably (C₁-C₆)alkyl. In another embodiment,alkyl is preferably (C₁-C₄)alkyl.

In an embodiment where the alkyl group is unsaturated, the alkyl is analkenyl group or an alkynyl group. Alkenyl can be, for example, vinyl,1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl,2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,4-hexenyl, or 5-hexenyl. The alkenyl can be unsubstituted orsubstituted. Alkynyl can be, for example, ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 1-octynyl, and the like. The alkynyl can be unsubstituted orsubstituted.

The term “aryl” refers to an aromatic hydrocarbon derived from a parentaromatic ring system. The aryl can be linked to another group at asaturated or unsaturated carbon atom of the parent ring system. The arylgroup can have 6 to about 14 carbon atoms. The aryl group can have asingle ring (e.g., phenyl) or multiple condensed (fused) rings, whereinat least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl,fluorenyl, or anthryl). Typical aryl groups include, but are not limitedto, radicals derived from benzene, naphthalene, anthracene, biphenyl,and the like. The aryl can be unsubstituted or substituted as describedherein. The term “halo” refers to fluoro, chloro, bromo, and iodo.Similarly, the term “halogen” refers to fluorine, chlorine, bromine, andiodine.

The term “substituted” is intended to indicate that one or more (e.g.,1, 2, 3, 4, or 5; in some embodiments 1, 2, or 3; and in otherembodiments 1 or 2) hydrogen atoms on the group indicated in theexpression using “substituted” is replaced with a selection from thesubstituents described hereinbelow, or with a suitable group known tothose of skill in the art, provided that the indicated substitutedatom's normal valency is not exceeded, and that the substitution resultsin a stable compound. Suitable substituent groups include, e.g., alkyl,alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl,heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino,alkylamino, dialkylamino, trifluoromethylthio, acylamino, nitro,difluoromethyl, trifluoromethyl, trifluoromethoxy, carboxy,carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, andcyano. The suitable substituent groups can also include, e.g., —X, —R,—OR, —SR, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NRR, —S(═O)₂OH, —S(═O)R,—S(═O)₂R, —O S(═O)₂OR, —S(═O)₂NR, —OP(═O)(OR)₂, —P(═O)(OR)₂,—P(═O)(OH)₂, —C(═O)R, —C(═O)X, —C(S)R, —C(O)OR, —C(S)OR, —C(O)SR,—C(S)SR, —C(O)NRR, —C(S)NRR, —C(NR)NRR, where each X is independently ahalogen (“halo”): F, Cl, Br, or I; and each R is independently H, alkyl,aryl, heterocycle, or a protecting group; or cations or anions thereof.As would be readily understood by one skilled in the art, when asubstituent is keto (i.e., ═O) or thioxo (i.e., ═S), or the like, thentwo hydrogen atoms on the substituted atom are replaced.

As to any of the above groups that contain one or more substituents, itis understood, of course, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds.

The term “diacid” refers to any group that contains two carboxylic acid(—C(═O)OH) groups. The diacid can be an aliphatic dicarboxylic acid oran aromatic dicarboxylic acid. An aliphatic dicarboxylic acid is anyalkyl group that is substituted with two (or more) carboxylic acidgroups. An aromatic dicarboxylic acid is any compound that contains anat least one aryl group and two (or more) carboxylic acids. The twocarboxylic acid groups can be on the same aryl group or they can be ondifferent aryl groups. When the two carboxylic acid groups are ondifferent aryl groups, the aryl groups can be linked by a single bond,or then can be linked by other groups, for example, an alkyl group. Thealkyl group linking the aryl groups can be optionally substituted andoptionally interrupted between carbons with other groups as definedherein.

The term “polymer” refers to a molecule of one or more repeatingmonomeric residue units covalently bonded together by one or morerepeating chemical functional groups. The term includes all polymericforms such as linear, branched, star, random, block, graft and the like.It includes homopolymers formed from a single monomer, copolymers formedfrom two or more monomers, terpolymers formed from three or morepolymers and other polymers formed from more than three monomers.Differing forms of a polymer may also have more than one repeating,covalently bonded functional group.

The term “polyanhydride” refers to a polymer that is derived from thecondensation of carboxylic acids or carboxylic acid derivatives suchthat repeating units of the resulting polymer are linked by anhydride(—C(═O)—O—C(═O)—) groups. Polyanhydrides can be prepared by condensingdiacids or by condensing anhydride prepolymers, as described herein.

The term “carboxylic anhydride” refers to a compound that contains ananhydride (—C(═O)—O—C(═O)—) group. A carboxylic anhydride typicallycontains only one anhydride group per molecule. Carboxylic anhydridescan be formed by the condensation of two carboxylic acids. Carboxylicanhydrides that can be used in conjunction with the methods describedherein include bis-alkyl carboxylic anhydrides, bis-aryl carboxylicanhydrides, and mixed anhydrides. Examples include, but are not limitedto acetic anhydride, trifluoroacetic anhydride, and benzoic anhydride.Mixed anhydrides can also be employed, such as acetic benzoic anhydride,which is the condensation product of acetic acid and benzoic acid.

As used herein, an “acyl” group is a group, such as a (C₁-C₄)alkylgroup, that terminates in a carbonyl radical at its point of attachmentto another group. An “acyloxy” group is a substituent, such as a(C₁-C₄)alkyl group, that terminates in a carboxyl radical at its pointof attachment to another group.

The term “acylated” refers to the conversion of a hydroxyl group into anacyloxy group. Acylation can be carried out by contacting a hydroxylgroup or hydroxyl-containing group with a carboxylic anhydride.

As used herein, a “prepolymer” is a monomer, oligomer, or mixturethereof that can be converted into a polymer (e.g., a longer chainpolyanhydride). Diacid prepolymers are typically acylated on theirterminal carboxy groups. A prepolymer can be, for example, abis(carboxylic acid acetyl ester), or an anhydride oligomer thereof. Insome embodiments, a prepolymer can be a1,ω-(4-acetoxycarbonylphenoxy)alkane, or an anhydride oligomer thereof.The phenoxy group of the 1,ω-(4-acetoxycarbonylphenoxy)alkane can haveortho, meta, or para substitution patterns.

As used herein, a “homopolymer” is a polymer that is made up ofrepeating units of one type of monomer. A “copolymer” is a polymer thatis made up of repeating units of two or more different types ofmonomers. In a random copolymer, the organization of the repeating unitsis random.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo. For example, amicrobe can be killed or inhibited when contacted with an antimicrobialagent.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect. Forexample, an amount effective can be an amount effective to reduce theprogression or severity of the condition or symptoms being treated.Determination of a therapeutically effective amount is well within thecapacity of persons skilled in the art. The term “effective amount” isintended to include an amount of a compound described herein, or anamount of a combination of compounds described herein, e.g., to treat orprevent a disease or disorder, or to treat the symptoms of the diseaseor disorder, in a host. Thus, an “effective amount” generally means anamount that provides the desired effect.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” extend to prophylaxis andinclude prevent, prevention, preventing, lowering, stopping or reversingthe progression or severity of the condition or symptoms being treated.As such, the term “treatment” includes both medical, therapeutic, and/orprophylactic administration, as appropriate.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

The particles described herein can encapsulate a variety of types ofcargo by incorporating the cargo molecules into the polyanhydride matrixof the particles. The particles can readily incorporate two or moredifferent types of active agents. Co-agents and/or additives such asdyes and radioactive nuclei may be included in the particles fordiagnostic purposes. Other additives can include compounds such asbacillomycin, which can enhance the activity of the active agent.Additionally, bacillomycin can be added to the particle formulation suchthat it is included inside the particles with a primary active agent,and outside the particle, in the pharmaceutical solution or linked tothe particle covalently by a linker such as PEG.

Accordingly, the particles can be loaded with a variety of differentactive agents. The term “active agent” (and its equivalents “agent,”“drug,” “bioactive agent,” “medicament” and “pharmaceutical”) isintended to have the broadest meaning and includes at least one of anytherapeutic, prophylactic, pharmacological or physiological activesubstance, cosmetic and personal care preparations, and mixturesthereof, which is delivered to an animal or plant to produce a desired,usually beneficial, effect. More specifically, any active agent that iscapable of producing a pharmacological response, localized or systemic,irrespective of whether therapeutic, diagnostic, cosmetic orprophylactic in nature, is within the contemplation of the invention.Bioactive agents such as pesticides, insect repellents, sun screens,cosmetic agents, and the like may be encapsulated by the particles.

It should be noted that the drugs and/or bioactive agents may be usedsingularly or as a mixture of two or more such agents, and in amountssufficient to prevent, cure, diagnose or treat a disease or othercondition, as the case may be. The drugs and mixtures thereof can bepresent in the composition in different forms, depending on which formyields the optimum delivery characteristics. Thus, in the case of drugs,the drug can be in its free base or acid form, or in the form of salts,esters, amides, prodrugs, enantiomers or mixtures thereof, or any otherpharmacologically acceptable derivatives, or as components of molecularcomplexes.

In various embodiments, the active agent can be, for example, anantimicrobial agent. The term “antimicrobial agent” refers to bioactivemolecules that kill or inhibit the growth or replication of bacteria,fungi, algae, or other pathogenic organisms, such as tuberculosis.Examples of drugs and antimicrobial agents that can be encapsulated inthe particles described herein include the generic and specific agentslisted at paragraphs [0057] to [0342] of U.S. Patent Publication No.2006/0078604 (Kanios et al.), which paragraphs are incorporated hereinby reference.

Additional examples of antimicrobial agents include sulfonamides,beta-lactams including penicillin, cephalosporin, and carbepenems,aminoglycosides, quinolones, and oxazolidinones, and metals such ascopper, iron, aluminum, zinc, gold, compound and ions thereof, andvarious combinations thereof. Other agents that can be included in thepolyanhydride particles include lipopolysaccharides (LPS),polyguanidines (CPG), bacterial lysates, such as material from a slurryof heat killed Brucella, e.g., to form a vaccine, and multi kDaproteins, such as defensins (cysteine-rich cationic proteins comprisingabout 18-45 amino acids).

The term “microbial infection” refers to an infection in an animalcaused by the proliferation of a microbe (a “microorganism”) in theanimal or within cells or tissue of the animal. The microorganisms canbe unicellular or members of a colony of cellular organisms. Examples ofmicrobes include bacteria, fungi, archaea, and protists.

Specific values listed herein for radicals, substituents, ranges, andother described values are for illustration only; they do not excludeother recited values or other values within defined ranges for radicalsand substituents in various embodiments. In other embodiments, anyrecited value or range may be excluded from the scope of an embodiment.

Polyanhydride Prepolymers, Polymers, and Synthesis Thereof.

The polyanhydrides used to prepare the particles of the invention can beprepared as described herein or by methods known to those of skill inthe art. Commercial diacids can be used as precursors for preparingprepolymers and the polyanhydrides. Techniques well known to those ofskill in the art can also be used to prepare diacids for prepolymer andpolyanhydride preparation. Many of these known techniques are elaboratedin Compendium of Organic Synthetic Methods (John Wiley & Sons, NewYork), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade,Jr., 1984; and Vol. 6, Michael B. Smith; as well as in March, J.,Advanced Organic Chemistry, 3^(rd) Ed., (John Wiley & Sons, New York,1985), Comprehensive Organic Synthesis; Selectivity, Strategy &Efficiency in Modern Organic Chemistry, in 9 Volumes, Barry M. Trost,Ed.-in-Chief (Pergamon Press, New York, 1993 printing), and Richard. C.Larock, Comprehensive Organic Transformations, 2^(nd) Ed., (Wiley-VCH,New York, 1999). A number of examples of methods for the preparation ofpolyanhydrides are provided below.

A wide range of suitable diacids can be employed to preparepolyanhydrides. The diacid can be a diacid-substituted straight orbranched chain alkane that is optionally interrupted by about one toabout five -Ph-, —O—, —CH═CH—, and/or —N(R)— groups wherein R is H,phenyl, benzyl, or (C₁-C₆)alkyl. In one embodiment, the alkane of thediacid can be C₂-C₁₂(alkyl). In another embodiment, the alkane can beC₄-C₈(alkyl). Additionally, the alkane group of the diacid can beoptionally interrupted by about 1 to about 12 —OCH₂CH₂O— groups, forexample, a poly(ethylene glycol) segment. The alkane group can also beoptionally substituted with one, two, or three (C₁-C₆)alkyl,(C₁-C₆)alkenyl, trifluoromethyl, trifluoromethoxy, or oxo groups; orcombinations thereof.

In one embodiment, a prepolymer can be prepared as illustrated in Scheme1.

wherein “organic group” is any organic group that can link twocarboxylic acid moieties, R is alkyl or aryl, and n is 1 to about 12.Examples of suitable organic groups include, but are not limited to,C₂-C₁₂(alkyl) groups, -PhO—C₂-C₁₂(alkyl)-OPh- groups, and PEG groupshaving 1 to about 12 PEG units, such as a 3,6-dioxaoctane group. A molarexcess of the carboxylic anhydride can be employed. About 2 to about 30molar equivalents of the carboxylic anhydride can be used.Alternatively, about 5 to about 20 molar equivalents of the carboxylicanhydride can be used. In one embodiment, 6 molar equivalents of thecarboxylic anhydride are employed. In another embodiment, 18 molarequivalents of the carboxylic anhydride are employed. The carboxylicanhydride can be, for example, acetic anhydride, trifluoroaceticanhydride, benzoic anhydride, combinations thereof, and/or derivativesthereof.

A prepolymer can also be prepared as illustrated in Scheme 2.

wherein n is 1 to about 12. Other carboxylic anhydrides can be used toform the end groups of the prepolymer, such as, but not limited to,benzoic anhydride. The central aliphatic group can optionally besubstituted or interrupted as described herein.

The diacid can also be a 1,ω-bis(carboxy)alkane. As would be recognizedby one skilled in the art, alternative nomenclature for a1,ω-bis(carboxy)alkane is a 1,ω-alkanedioic acid that has two additionalcarbons in the alkane moiety compared to the correspondingbis(carboxy)alkane.

A prepolymer can also be prepared as illustrated in Scheme 3.

wherein n is 1 to about 12. Carboxylic anhydrides other than aceticanhydride can be used to form the end groups of the prepolymer. Thecentral aliphatic group, the aryl groups, or both, can optionally besubstituted, in any combination. The central aliphatic group can also beinterrupted as described herein.

Accordingly, the diacid can be two aryl groups that are each substitutedwith a carboxy group wherein the aryl groups are linked by a straight orbranched chain alkane that is optionally interrupted by about one toabout five -Ph-, —O—, —CH═CH—, and/or —N(R)— groups wherein R is H,phenyl, benzyl, or (C₁-C₆)alkyl. In some embodiments, one or both of thearyl groups can be omitted and the carboxy groups are linked by thealkyl chain. In one embodiment, the alkane can be C₂-C₁₂(alkyl). Inanother embodiment, the alkane can be C₄-C₈(alkyl). In anotherembodiment, the alkane can be one or more PEG groups. Additionally, thealkane group linking the carboxylic acid-substituted aryl groups can beoptionally interrupted by 1 to about 12 —OCH₂CH₂O— groups, for example,a poly(ethylene glycol) segment. The alkane group linking the carboxylicacid-substituted aryl groups can also be optionally substituted withone, two, or three (C₁-C₆)alkyl, (C₁-C₆)alkenyl, trifluoromethyl,trifluoromethoxy, or oxo groups; or combinations thereof.

The diacid can be a 1,ω-bis(4-carboxyphenoxy)alkane. In one embodiment,the alkane is a (C₂-C₁₀)alkane. In another embodiment, the alkane can bea C₄-C₈(alkyl). In certain specific embodiments, alkane can be ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, and branched isomersthereof. In one embodiment, the diacid is a1,6-bis(4-carboxyphenoxy)hexane. In another embodiment, the diacid is a1,6-bis(carboxy)octane. In another embodiment, the diacid can be a1,8-bis(carboxyphenoxy)-3,6-dioxaoctane. Mixtures of any of thesediacids can be used in conjunction with the microwave facilitatedmethods described herein.

Polyanhydrides.

Polyanhydrides can be prepared by condensation methods known in the artor by irradiating a prepolymer with a sufficient amount of microwaveirradiation to polymerize the prepolymer. A sufficient amount ofmicrowave radiation can typically be generated by a conventionalmicrowave oven set to 1100 Watts for about 1 to about 30 minutes. Moreoften, a sufficient amount of microwave radiation can be generated inabout 1 to about 20 minutes. The resulting polyanhydride can be ahomopolymer or a copolymer, depending on the nature of the prepolymercomposition used in the reaction.

A polyanhydride can also be prepared by forming a prepolymer in situfrom diacids. The diacids can be converted into prepolymers byirradiating diacids in the presence of a carboxylic anhydride. Theprepolymer can be prepared by, for example, by irradiating a mixture of(a) a carboxylic anhydride and (b) an aromatic dicarboxylic acid, analiphatic dicarboxylic acid, or a mixture thereof, with an amount ofmicrowave radiation effective to form the prepolymer. One suitablecarboxylic anhydride is acetic anhydride. Other suitable carboxylicanhydrides include, for example, trifluoroacetic anhydride and benzoicanhydride.

The terminal groups of polyanhydrides prepared according to the methodsdescribed herein will typically have terminal acyl groups. It ispossible for some hydrolysis of the polyanhydrides to occur during thereaction or during the isolation of the polyanhydride. Thus, someterminal groups of such polyanhydrides can be carboxylic acid groups.Accordingly, the methods of the invention include the preparation ofpolyanhydrides that terminate in acyl groups, carboxylic acid groups, orcombinations thereof.

The polyanhydride can be prepared, for example, as illustrated in Scheme4.

where “organic group” is any organic group that links two carboxylicacid moieties, R is alkyl or aryl, n is 1 to about 12, and m is about 5to about 200.

The polyanhydride can also be prepared as illustrated in Scheme 5.

where n is 1 to about 12 and m is about 5 to about 200. In otherembodiments, m can be about 10 to about 100, or about 10 to about 50. Aswould be understood by one skilled in the art, the value of m willtypically be larger than the value of n. End groups other than acetatecan be used and the central aliphatic group can be optionallysubstituted or optionally interrupted (e.g., as for PEG groups), orboth, as described herein.

The polyanhydride can also be prepared as illustrated in Scheme 6.

wherein n is 1 to about 12 and m is about 5 to about 100. In otherembodiments, m can be about 10 to about 50, or about 15 to about 35. Endgroups other than acetate can be used and the central aliphatic group,the aryl groups, or both, can optionally be substituted, in anycombination. The central aliphatic group can also be optionallyinterrupted as described herein.Polyanhydride Polymers for Preparation of Microparticles andNanoparticles.

A method for preparing the polyanhydride microparticles or nanoparticlesincludes irradiating one or more diacids, wherein the one or morediacids include an aromatic dicarboxylic acid, an aliphatic dicarboxylicacid, or a mixture thereof, with microwave radiation in the presence ofa carboxylic anhydride so as to acylate one or more diacids to yield atleast one prepolymer; and irradiating the prepolymer with microwaveradiation so as to polymerize said prepolymer to yield thepolyanhydride, as a homopolymer or a copolymer.

The prepolymers can be made up of dicarboxylic acids (“diacids”) thatare acylated at both acid moieties. A prepolymer can be a singleacylated diacid unit (monomer), or it can have up to about 12 condenseddiacid units. A mixture of different diacids can be employed. Themixture of diacids can yield a random copolymer. The one or more diacidscan include a diacid-substituted C₂-C₁₂ straight or branched chainalkane that is optionally interrupted by about 1 to about 5 -Ph-, —O—,—CH═CH—, and/or —N(R)— groups wherein R is H, phenyl, benzyl, or(C₁-C₆)alkyl. The one or more diacids can also be optionally interruptedby about 1 to about 12-OCH₂CH₂O— groups. The one or more diacids canalso be optionally substituted with 1, 2, or 3 trifluoromethyl,trifluoromethoxy, (C₁-C₆)alkyl, (C₁-C₆)alkenyl, or oxo groups, orcombinations thereof.

The at least one diacid can be a 1,ω-bis(carboxy)alkane. The at leastone diacid can also be a 1,ω-bis(4-carboxyphenoxy)alkane. The alkane canbe, for example, a (C₃-C₈)alkane. Specific examples of the alkaneinclude hexane and octane. The diacid can be1,6-bis(4-carboxyphenoxy)hexane. Alternatively, the diacid can be1,6-bis(carboxy)octane (sebacic acid). The at least one prepolymer canalso include a bis(carboxylic acid acetyl ester), or an anhydrideoligomer thereof. The at least one prepolymer can also include a1,ω-(4-acetoxycarbonylphenoxy)alkane, or an anhydride oligomer thereof,or a 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane, or an anhydride oligomerthereof.

The carboxylic anhydride can be a bis-alkyl carboxylic anhydride, abis-aryl carboxylic anhydride, an alkyl-aryl carboxylic anhydride, or amixture thereof. The carboxylic anhydride can be, for example, aceticanhydride, trifluoroacetic anhydride, or benzoic anhydride. A molarexcess of the carboxylic anhydride can be employed. Excess carboxylicanhydride can be removed after the prepolymer has formed.

In various embodiments, the polymers of the microparticles and/ornanoparticles described herein can be poly-sebacic anhydrides (SA),poly-1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydrides, orpoly-1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) anhydrides. Inother embodiments, the polymers of the microparticles and/ornanoparticles described herein can be copolymers of sebacic anhydride(SA) and 1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride, or copolymersof 1,8-bis(carboxyphenoxy)-3,6-dioxaoctane (CPTEG) anhydride and1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride. The ratio of SA toCPH, or CPTEG to CPH, can be any integer from about 1:19 to about 19:1.An example of a structure of a SA:CPA copolymer is:

where each block (designated by a single or double bracket) includes anumber of repeating units sufficient to provide a polymer with an M_(n)of about 5,000 to about 50,000 g/mol, such as about 10,000 to about25,000 g/mol, or about 15,000 to about 20,000 g/mol. The anhydridecopolymer can be a block copolymer or a random copolymer, or acombination thereof. CPTEG:CPH copolymers can also be prepared to formpolymers where each block can include a number of repeating unitssufficient to provide a polymer with an M_(n) of about 5,000 to about50,000 g/mol, such as about 10,000 to about 25,000 g/mol, or about15,000 to about 20,000 g/mol.

The PA particles, or polyanhydride nanospheres (PANS), described hereincan be loaded with an effective amount of an antimicrobial agent. ThePANS have been loaded with doxycycline and numerous model agents. Thissuccessful loading indicates that any antimicrobial agent, includingboth hydrophilic and hydrophobic agents, and be effectively encapsulatedwithout agent deterioration. For example, PA particles (PANS) that haveencapsulated doxycycline with either 1.5% or 3% loading kill laboratoryand field strains of Brucella canis and laboratory strains ofEscherichia coli by agar disk diffusion assays. Once administered toinfected cells, the PANS can localize in intracellular compartments thatcontain the intracellular pathogen. This localization can occur withoutkilling the host cell that internalized the PANS.

Antimicrobial Polyanhydride Particles.

The polyanhydride particles can incorporate a wide variety of cargomolecules into the matrix of the polyanhydride polymers that make up theparticles. Several antibiotics tested in the particles include amikacin,streptomycin, doxycycline, erythromycin, gentamicin, isoniazid,rifampin, and ethambutol. Current loading percentage of antibiotics havebeen successful up to about 5-10% w/v of copolymer, and higher loadingpercentages, as high as 50%, can be achieved by techniques such assequential loading of additional active agent and anhydride polymers toform larger particles. The particles can readily be loaded with about 1μg to about 12 μg of an antimicrobial agent per particle.

Demonstration of Antimicrobial Activity.

To determine the antimicrobial activity of the PANS, the releasekinetics from encapsulated nanospheres can be determined by quantifyingthe amount of encapsulated antimicrobial agent, such as doxycycline,released from nanospheres. The chemical structure of releasedantimicrobial agent can be confirmed by analyzing for the presence ofderivative molecules by circular dichroism and MS-HPLC. Antimicrobialactivity for the released antimicrobial agent can also be confirmed.Results of this analysis can be used to determine the highest amount ofencapsulated antimicrobial agent that can be delivered while remainingbelow host cytotoxicity levels for released antimicrobial agent. Thisinformation allows for the determination of optimal antimicrobial agentloading to develop whole animal treatment protocols. In one specificembodiment, the Brucella infected cells can be infected with B.melitensis. The formulation can be used to eliminate B. melitensisresiding within macrohpages.

Pharmaceutical Formulations.

The compositions described herein, for example, the polyanhydrideparticles encapsulating antimicrobial agents, can be used to preparetherapeutic pharmaceutical compositions. The compositions describedherein can be formulated as pharmaceutical preparations and can beadministered to animal hosts, such as a mammalian host, for example ahuman patient. The preparation can be provided in a variety of forms.The forms can be specifically adapted to a chosen route ofadministration, e.g., oral or parenteral administration, intravenous,intramuscular, topical or subcutaneous administration, or administrationby inhalation.

The pharmaceutical compositions may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration,compositions can be enclosed in hard or soft shell gelatin capsules,compressed into tablets, or incorporated directly into the food of apatient's diet. Compositions may also be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations typically contain at least 0.1% ofactive compound. The percentage of the compositions and preparations canvary and may conveniently be from about 1% to about 25% of the weight ofa given unit dosage form. The amount of active compound in suchtherapeutically useful compositions is such that an effective dosagelevel can be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active composition, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound in the particle composition may be further incorporated intosustained-release preparations and devices.

The active agent, e.g., the antimicrobial agent, may be administered inthe polyanhydride particles intravenously or intraperitoneally byinfusion or injection. Solutions of the pharmaceutical compositions canbe prepared in water, optionally mixed with a nontoxic surfactant.Dispersions can be prepared in glycerol, liquid polyethylene glycols,triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil.Under ordinary conditions of storage and use, preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the compositions adapted for the extemporaneous preparationof sterile injectable or infusible solutions or dispersions. Theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thiomersal, and the like. In manycases, it may be advantageous to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of injectablecompositions can be brought about by agents delaying absorption, forexample, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating thecompositions in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparation caninclude vacuum drying and freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, compositions may be applied in pure form,e.g., in conjunction with a single carrier. However, it will generallybe desirable to administer the active agents in the particles to theskin as a composition or formulation, for example, in combination with adermatologically acceptable carrier formulation, such as a gel,ointment, lotion, foam, or cream.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, orwater-alcohol/glycol blends, in which the compositions can be dissolvedor dispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed using a pump-typeor aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of dermatological compositions fordelivering active agents to the skin are known to the art; for example,see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157), and Wortzman (U.S.Pat. No. 4,820,508). Such dermatological compositions can be used incombinations with the compositions described herein.

Useful dosages of the compositions described herein can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of a composition requiredfor use in treatment will vary not only with the particular active andencapsulating polymer, but also with the route of administration, thenature of the condition being treated, and the age and condition of thepatient, and will be ultimately at the discretion of an attendantphysician or clinician.

The compositions can be conveniently administered in a unit dosage form,for example, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m²,most conveniently, 50 to 500 mg/m² of active ingredient per unit dosageform. The desired dose may conveniently be presented in a single dose oras divided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The invention therefore provides therapeutic methods of treatingmicrobial infections in a mammal, which methods involve administering toa mammal having a microbial infection an effective amount of acomposition described herein. A mammal includes a primate, human,rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovineand the like.

The ability of a compositions of the invention to treat microbialinfections may be determined by using assays well known to the art. Forexample, the design of treatment protocols, toxicity evaluation, dataanalysis, quantification of cell kill, and the biological significanceof the use of screens are know.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Antibodies and Reagents for Fluorescence Microscopy

Primary antibodies used for immunofluorescence microscopy were asfollows: mouse anti-LAMP 1 monoclonal, anti-mannose-6-phosphate receptor(Iowa State Hybridoma), mouse anti-EEA1, anti-calnexin, anti-BiP/GRP74,anti-p115, anti-p230, anti-SRP54, anti-RabS (Transduction Laboratories),mouse anti-Transferrin Receptor (Molecular Probes), rabbit anti-Brucellaantibody (Difco), rabbit anti-E. coli antibody (Molecular Probes) andmouse/rabbit anti-Cathepsin D (Oncogene). Primary antibodies were usedroutinely at the concentration of 1/100 except for the following: mouseanti-LAMP-1, 1/20; and mouse anti-mannose-6-phosphate receptor, 1/5.Slow-fade and Prolong antifade mounting solution and Lysotracker RedDND-99 were purchased from Molecular Probes.

Example 1 Preparation of Polyanhydrides and Polyanhydride Nanospheres

Sebacic acid (99%), 4-hydroxybenzoic acid, 1-methyl-2-pyrrolidinoneanhydrous (99.5%), 1,6-dibromohexane (98.5%) andfluorescein-isothiocyanate-dextran (FITC-dextran) were purchased fromSigma-Aldrich (Milwaukee, Wis., USA), Other chemicals were purchasedfrom Fisher Scientific (Pittsburgh, Pa., USA) and used as received.

Synthesis of SA and CPH pre-polymers and copolymers was performed aspreviously described (M. J. Kipper et al., Biomaterials.23(22):4405-4412 (2002); E. Shen et al., J. Control. Release.82(1):115-125 (2002); A. Conix, Poly[1,3-bis(p-carboxyphenoxy)propaneanhydride], Macromolecular Synthesis, 2:95-98 (1966); and U.S. Pat. No.7,659,322 (Vogel et al); each incorporated herein by reference).

The resulting polymers were characterized using ¹H nuclear magneticresonance to verify polymer chemistry, gel permeation chromatography toanalyze molecular weight, and differential scanning calorimetry todetermine glass transition temperature and crystallinity. All propertiesevaluated showed that the synthesized polymers were within acceptedranges.

Nanosphere Fabrication and Characterization.

FITC-dextran loaded nanospheres were fabricated by polyanhydrideanti-solvent nanoencapsulation (PAN), similar to the method reported byMathiowitz et al. for poly (fumaric acid-co-sebacic acid) polymers (E.Mathiowitz et al., Nature, 386(6623):410-414 (1997)). Active agents canbe encapsulated into the nanospheres in a similar manner. Polymer (145.5mg) was dissolved in methylene chloride (5 mL) held at room temperaturefor poly(SA) and 20:80 CPH:SA, and at 0° C. for 50:50 CPH:SA.FITC-dextran (4.5 mg) was added to the polymer solution and homogenizedat 30,000 rpm for 30 seconds to create a suspension. Thepolymer/fluorescein solution was rapidly poured into a bath of petroleumether at an antisolvent to solvent ratio of 80:1 held at roomtemperature (˜23° C.) for poly(SA) and 20:80 CPHSA, and −40° C. for50:50 CPHSA (due to the lower glass transition temperature for 50:50CPH:SA (12)).

Polymer solubility changes due to the presence of anti-solvent causedspontaneous particle formation. These particles were removed from theanti-solvent by filtration (by aspiration using a Buechner funnel andWhatman #2 filter paper) and then dried overnight under vacuum. Theprocedure yielded a fine powder with at least 70% recovery. Thenanosphere morphology was investigated using scanning electronmicroscopy (JEOL 840A, JEOL Ltd., Tokyo, Japan). Particle diameter wasdetermined using quasi-elastic light scattering (Zetasizer Nano, MalvernInstruments Ltd., Worcester, UK).

Example 2 Polyanhydride Microspheres and Nanospheres

Existing Technology.

Particle encapsulated antibiotics for treating bacterial infections havebeen prepared using PLGA microspheres and nanospheres. These deliverysystems provide minimal, if any, therapeutic benefit. These poor resultsare magnified for chronic bacterial diseases such as Brucella andMycobacterium tuberculosis.

Poly(D,L-lactide-co-glycolide) (PLGA) microspheres and nanospheres havebeen prepared for certain drug delivery applications. PLGA microspheresand nanospheres are hydrophilic, and degrade by water hydrolyzeddegradation. The rate of degradation increases at acidic pH, and themechanism of erosion has been determined to be bulk erosion. Therelative degradation in tissues is rapid, taking only hours to a fewdays. For example, 95% of PLGA microspheres were degraded within oneweek (Lecaroz et al., Poly(D,L-lactide-coglycolide) particles containinggentamicin: pharmacokinetics and pharmacodynamics in Brucellamelitensis-infected mice. Antimicrob. Agents Chemother. 2007, 51:1185-1190). Lecaroz and coworkers cited some success in reducingbacteria within the spleens of mice infected with Brucella melitensis byreporting that the log # of bacteria were reduced from 6.73 to 5.29.However, the mice were still very much infected. A reduction of thismagnitude would not provide a suitable clinical treatment for humans.

TABLE 2 of Lecaroz, 2007. Protective effect of three doses of GEN-loadedmicroparticles against sublethal infection with Brucella melitensis 16Madministered intraperitoneally. Results for the following time afteradministration of last dose^(a): 1 wk 3 wk Spleen wt Log ReductionSpleen wt Log Reduction Treatment (g) CFU/spleen (log) (g) CFU/spleen(log) Untreated 0.87 ± 0.08 6.85 ± 0.17 0.00 0.93 ± 0.11 6.73 ± 0.180.00 Free GEN 0.86 ± 0.05 6.77 ± 0.12 0.08 0.83 ± 0.26 6.77 ± 0.12 0.04502H empty Mp^(b) 0.80 ± 0.05 6.83 ± 0.16 0.02 1.04 ± 0.14 6.87 ± 0.300.13 75:25H empty MP 0.78 ± 0.28 6.80 ± 0.12 0.05 1.16 ± 0.19 6.70 ±0.18 0.04 502H GEN MP 0.92 ± 0.07 6.44 ± 0.05 0.41** 1.12 ± 0.44 6.28 ±0.09 0.45 75:25H GEN MP 0.87 ± 0.16 6.13 ± 0.08 0.72** 0.70 ± 0.36 5.29± 1.58 1.45** ^(a)Groups of six mice each were infectedintraperitoneally with B. meditensis 16M (1 × 10⁵ CFU/mouse). After 2weeks, the animals received three doses of free or encapsulated GEN (1.5mg/kg). At 1 and 3 weeks after administration of the last dose, theanimals were killed. **, P < 0.01 (Mann-Whitney U test). ^(b)MP,microsphere.

As can be seen from the Lacaroz Table 2, the Lacaroz procedure merelyreduced the number of cell forming units and did not treat the bacterialinfection to any appreciative or clinically relevant extent.

Examples of polyanhydride microspheres and nanospheres of the invention.In various embodiments, polyanhydride microspheres and nanospheres canbe copolymerized particles (“copolymer”) of a hydrophobic monomer (CPH)and a hydrophilic monomer (either SA or CPTEG). For example, certainpolyanhydride nanospheres described herein are based on the monomerssebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and/or1,8-bis(p-carboxyphenoxy)3,6-dioxaoctane (CPTEG). These polyanhydrideparticles can be used for applications such as drug delivery and/orvaccine delivery.

The polyanhydride particles that include CPH have hydrophobicproperties, they resists hydrolytic degradation in vivo, and they resistdegradation at acidic pH. They also degrade slowly, and the degradationoccurs by a surface erosion mechanism (vs. bulk erosion by PLGA).Encapsulated cargo can thus be slowly released during surface erosion.Very little leaching of cargo from intact particles occurs.

The relative degradation in tissues is weeks to months, depending on thesize and specific polyanhydride used to prepare the particles. Spleensand livers of Mice that have been injected with small numbers ofparticles had high remaining levels after 1 week (FIG. 1). In dendriticcells and monocytes, two hour counts of particles internalized/dendriticcell are shown in FIG. 1. In dendritic cells, 48 hour counts ofparticles internalized/dendritic cell are shown in FIG. 2. FIG. 3illustrates intracellular compartment localized to lysosomes by LAMP1staining. FIG. 4 illustrates intracellular localization of Brucellaabortus within monocytes (same lineage as dendritic cells). This figuredemonstrges that particles and Brucella reside within the sameintracellular compartment.

Example 3 Intracellular Survival and Replication of B. abortus Strainsin THP-1 Cells

THP-1 cells used for the evaluation of the intracellular survival andreplication profiles of the B. abortus strains were plated into 96 wellflat bottom tissue culture plates. Bacterial suspensions were preparedgenerated by scraping 48 hour cultures of the B. abortus strains grownon BA into screw cap microfuge tubes containing PBS. Pellets of bacteriawere re-suspended by vigorous vortexing and numbers of bacteria presentin the suspensions were determined by OD₆₀₀ measurements. Opsonizationtook place within these dilute suspensions containing either rabbitanti-Brucella (Difco) or murine complement component (C3) isolated freshfrom non-infected mice. Concentrations of antibody necessary to mediateopsonization without agglutinating the bacterial suspensions wereachieved using antibody dilutions ranging from 1/2000 to 1/5000.

Bacterial cell suspensions and antisera were incubated together eitherat 37° C. in a shaking water bath for 20 minutes or at room temperaturefor 30 minutes followed by brief vortexing. Suspensions of opsonizedbacteria were added to monocyte monolayers at a multiplicity ofinfection (bacteria:monocyte ratio) 20:1 for B. abortus 2308. Tissueculture plates were gently agitated by hand then centrifuged at 4° C.for 10 minutes at 270×g. Monolayers were washed gently with cold PBS toremove non-adherent bacteria then incubated in fresh medium for 20minutes at 37° C. with 5% CO₂ to allow for phagocytosis of adherentbacteria. Monolayers were washed 3 times with PBS to remove anyremaining non-adherent bacteria.

Fresh media containing 100 μg/mL gentamicin was added following the lastwash step to kill adherent, extracellular bacteria. For experimentslasting longer than 2 hours, the 100 μg/mL gentamicin supplementedmedium was replaced with medium containing 10 μg/mL gentamicin after 1hour and remained in this medium for the duration of the experiment.

Viability of intracellular Brucella was determined by lysing monocyteswith 0.1% deoxycholate, diluting suspensions in PBS and plating aliquotsin triplicate on BA medium (Bellaire, Elzer et al. 1999). Percentsurvival of bacteria at 24 and 48 hours was calculated based on thenumber of internalized bacteria detected at 1 hour post infection, whichrepresents 100% of internalized bacteria. Statistical comparisons weremade using Student's t-test. Host cell cytotoxicity assays for culturedcells can be recorded using the MTT assay for quantitative measurementsand by propidium iodide exclusion for microscopy purposes.

FIG. 5 illustrates quantifying the intracellular stability ofnanospheres using morphometric image analysis. Images ofFITC-encapsulated nanospheres were captured using 40× objective andprocessed using constant values for camera exposure and imagethresholding throughout the experiment. Images of Lamp1 and nucleistained DC's incubated with nanospheres at indicated times werebackground subtracted to generate binary equivalent image (FITC Binary)to perform particle analysis using ImageJ v1.42 software (bar=5 μm).FIG. 6 illustrates binary particle counts and pixel area results asdetermined from FIGS. 1 and 2. Binary particle counts and pixel arearesults were averaged from at least 15 separate fields of view for eachtime point and particle composition.

Using information regarding effective concentrations determined above,mice can be experimentally infected with virulent Brucella abortusstrain 2308 (example of chronic infection shown in FIG. 7) and treatedwith PANS by single dose (˜10 μg/kg) i.v. tail vein injection at 4 weekspost infection. The status of the chronic infection within BALB/c micecan be measured by harvesting the spleens and livers of mice at 1 weekintervals after infection, the tissues can be homogenized in PBS,serially diluted and plated on Schaedler agar plates supplemented with5% bovine blood.

BABL/c mice were chosen because they are regarded as being moresusceptible to Brucella infection, where the bacterial load in thespleen and liver persists at a much higher level than seen in Blk6 mice(Baldwin and Parent 2002). The typical chronic infection includes the“plateau phase” reached at week 2 and extends to 12 weeks and beyond(FIG. 7). Mice are infected i.p. with 1×10⁵ CFU in PBS suspension.

FIG. 7 illustrates an experimental infection of Blk6 mice as indicatedabove. The representative experiment illustrates the establishment ofinfection at 1 week and how it is followed by maintenance of the chronicinfection in the spleens of mice infected i.p. with Brucella. CFU ofBrucella recovered from the spleens of these animals are shown as anaverage of 5 mice per group per time point. Student's T-test indicatesthat the KO mouse strain has fewer

Brucella, illustrating how changes in Brucella colonization can bequantified between different treatment groups. Ad hoc statisticalanalysis can also include one-way Anova to detect drug specific changesover the entire course of the experiment.

Example 4 Stimulation of Monocytes with Interferon-γ (INF-γ) Inhibitsthe Intracellular Replication of Brucella

The intracellular trafficking and fate of PANS can overlap considerablywith the location of Brucella containing vesicles in infectedmacrophages. This can be confirmed using immunofluorescence microscopyto examine the effects of particle chemistry (polymer composition) onPANS internalization, intracellular trafficking, and intracellularcompartmentalization. To understand how variations in nanoparticlechemistry impact in vivo anti-Brucella activity, microscopic experimentscan be carried out to visualize how PANS of different copolymerformulations interact with Brucella containing replicative vesicleswithin murine and human macrophage cell lines.

In Vitro Assessments.

In vitro bacterial survival assays can be carried out, where the numberof viable intracellular Brucella recovered from monocytes is quantifiedby colony plating. FIG. 8 reveals the typical survival curve of Brucellawithin monocytes where a significant number of bacteria are killed overthe first 24 hour period, while the bacteria continue to replicate overthe second 24 hour period. In addition to the human THP-1 monocytic cellline used for bacterial survival assays, parallel experiments can beperformed with the murine cell line J774A. Both of these cell lines havebeen used numerous times in Brucella intracellular viability studies andhave been shown to accurately model the infection as compared to primarycells (Rittig, Alvarez-Martinez et al. 2001; Bellaire, Roop et al.2005).

Experiments using J774A cells follow the protocol outlined in Example 3for THP-1 cells. Results from experiments with THP-1 cells clearlydemonstrate that entry of the bacteria and replication within cells, asmeasured over the course of the 48 hours of infection, are reduced forcultures treated with both soluble doxycycline and doxycyclineencapsulated PANS (equivalent of 10 μg/mL for both groups), however thedoxycycline encapsulated PANS was significantly more effective.

Example 5 Nanosphere Degradation in Dendritic Cells

Nanosphere degradation in dendritic cells is a strong function of thepolymer structure and properties of the nanosphere material. Thenanospheres described herein elicits novel phagocytic response fromdendritic cells due to the chemical composition of the nanospheres, suchas increased hydrophobicity compared to known treatment methods.Reference can again be made to FIG. 5, which illustrates quantifying theintracellular stability of nanospheres using morphometric imageanalysis. Images of FITC-encapsulated nanospheres were captured using40× objective and processed using constant values for camera exposureand image thresholding throughout the experiment. Images of Lamp1 andnuclei stained dendritic cells incubated with nanospheres at indicatedtimes were background subtracted to generate binary equivalent image(FITC Binary) to perform particle analysis using ImageJ v1.42 software(bar=5 μm). Also, FIG. 6 illustrates binary particle counts and pixelarea results as determined from FIG. 1. Binary particle counts and pixelarea results were averaged from at least 15 separate fields of view foreach time point and particle composition.

Immature dendritic cells internalize (<2 μm) particles by phagocytosis.The amount of nanoparticles internalized is dramatically lower thanobserved for PStB polymerized actin localized with the phagosomal cup.Polymerized actin is retained around newly formed phagosomes containingPANS compared to PStB. To analyze the cellular mechanisms drivingparticle uptake, actin polymerization, lipid raft aggregation, andclathrin deposition can be inhibited.

To determine if nanosphere hydrophobicity affects the fate of particleswithin dendritic cells (DCs), equivalent amounts of FITC-dextran loaded20:80 CPH:SA and 50:50 CPH:SA nanospheres were incubated with immatureC3H DCs for 30 minutes, then washed to remove extracellular nanospheres.Cultures were incubated with fresh media for an additional 2 or 48 hoursto visualize particle uptake (2 hours) and intracellular stability (48hours) using immunofluorescence microscopy.

FIG. 9 illustrates the enhanced uptake of soluble Eα-RFP antigen bymonocytes after co-incubation with polyanhydride nanospheres for 2hours. Data demonstrated that the poly(SA) nanospheres enhanced antigeninternalization more readily than did 20:80 CPH:SA followed by 50:50CPH:SA. Representative epifluorescent images were captured and processedusing identical exposure and ImageJ software settings. Adjacent bargraphs summarize the average amount of RFP detected per cell. Pixelareas within each image correspond to relative intensity of RFP signaldetected inside cells. Values from three randomly selected fields ofview were used to calculate averages and standard deviation. Scale bar=5μm.

Polyanhydride Nanospheres Enhance Antigen Internalization.

It was demonstrated that polymer chemistry significantly influences theuptake of a model antigen (Eα tagged with red fluorescent protein (RFP),henceforth referred to as Eα-RFP), by THP-1 human monocytic cells. TheTHP-1 cells were co-incubated with blank nanospheres (poly(SA), 20:80CPH:SA, or 50:50 CPH:SA) and soluble Eα-RFP.

In order to evaluate phagocytic processes, the nanospheres wereco-incubated with the THP-1 cells for 30 minutes. Cultures were washedand the cells were placed back in the incubator for 2 hours prior toanalysis. In order to evaluate endocytic processes, the nanospheres wereco-incubated with the THP-1 cells for 6 hours. Cultures were washed andthe cells were placed back in the incubator for 48 hours prior toanalysis. The cells were fixed and visualized by epifluorescencemicroscopy employing TRITC/rhodamine filter set with 510-560 nmexcitation and 575-645 nm emission. Image black levels for the RFPprotein were set using cells not incubated with RFP. Exposure times forRFP detection were kept constant throughout the experimental groups tofacilitate accurate comparative analysis. Bar graphs of the relativepixel intensity of internalized RFP were calculated from RAW-RFP imagesusing the ImageRplugin/histogram function.

These bar graphs reveal the relative pixel intensity of the RFP proteindetected in the presence and absence of nanospheres. Representativephotomicrographs and bar graphs summarizing cell associated RFP data areprovided in FIG. 9. Comparisons among the three chemistries reveal thatafter 2 hours of co-incubation, all three chemistries dramaticallyincreased the amount of soluble antigen internalized by monocytes.

A potential mechanism for the increase in uptake stimulated by thenanospheres is that the protein itself is able to adsorb on the surfaceof nanospheres that are then subsequently internalized by the APC.However, control experiments failed to detect soluble RFP adsorbing ontoFITC-labeled nanospheres and culture conditions include ample amounts ofserum proteins present in the 10% fetal bovine serum supplementedmedium. This data demonstrates that the chemistry of the polyanhydridenanospheres influences the ability of APCs to internalize solubleantigen.

These studies indicate that the chemistry of the carrier has asignificant effect on protein stability. Ova has a tendency to formmoisture-induced covalent aggregates, which is shown by the presence ofcharacteristic bands between 54 and 97 kDa (lane 2), in addition to themajor Ova band at 48 kDa (data not shown). Strongly antigenicallyreactive bands are visible for both states, which was comparable to theunencapsulated Ova, for the protein released from each of theCPTEG-containing polymers (lanes 6-8), indicating that the antigenicepitopes of Ova were conserved.

This data indicate that the primary structure of Ova was not altered byencapsulation and release from the CPTEG-containing polymers.Encapsulation and release from 50:50 CPH:SA microspheres also appearedto preserve antigenic epitopes of the 45 kDa Ova, however, only faintbands for the aggregated forms of Ova were detectable. Poly(SA) and20:80 CPH:SA appeared to degrade Ova below the limit of detection byimmunoblot analysis; also showed a similar effect. Even thoughequivalent amounts of protein were loaded on to each SDS-PAGE gel, thedata indicated that polyclonal antibody was unable to detect Ova thathad been encapsulated into poly(SA) and 20:80 CPH:SA copolymer,indicating that the protein had been degraded.

Based on the immunoblot analysis, there was less degradation of Ova thatwas encapsulated into the 50:50 CPH:SA microspheres and the antigenicrecognition was similar to that of un-encapsulated Ova. It is likelythat the observed degradation was related to the acidic microenvironmentresulting from the degradation of poly(SA) and 20:80 CPH:SA. On theother hand, epitope integrity was better maintained in CPTEG-containingmicrospheres regardless of composition or fabrication method. This islikely due to the amphiphilic nature of the CPTEG-containing copolymersand the lower acidity of the resultant degradation products aspreviously reported.

Example 6 Model System for Treatment of Human Tuberculosis Infections

A new tractable system to model human tuberculosis (TB) infections hasbeen developed. This system uses the small laboratory fish, Japanesemedaka (Oryzias latipes), as the host and employs Mycobacterium marinumas the pathogen. Both pathogen and host genomes have been sequenced andare genetically tractable. Unlike M. tuberculosis, this surrogatepathogen can be easily manipulated, has good molecular genetic systems,grows fast and represents negligible BSL2 risks to laboratory workers.

Analogous to human TB, it was discovered that M. marinum mounts alife-long chronic disease in medaka without obvious overt symptoms inmost individuals. Histopathology analysis revealed that target organsincluding the spleen, kidney and liver were infected, and granulomaswere present in these tissues, the hallmark lesions of human TB. Mutantstrains of M. marinum have been successfully engineered with inactivatedgenes that are either known or suspected to be virulence genes in M.tuberculosis. Reduced colonization and spread was observed for some ofM. marinum mutants in fish. Detailed analyses of these and other mutantscan be extended by performing infections with lines of medaka that aredevoid of pigments.

To better model “fish TB” and monitor bacterial colonization, a mutantcolony of medaka was established that is devoid of most pigments. Thesetranslucent lines of fish, referred to as “See Through Medaka (ST)”,permit the viewing of all major organs in live animals (Broussard andEnnis, 2007). The ST fish were experimentally infected with wild-type M.marinum expressing either green fluorescent protein (gfp) or redfluorescent proteins (rfp). Real-time bacterial spread and colonizationof organs by either gfp- or rfp-expressing bacteria were monitored inreal time.

Bacterial colonization levels of target organs are convenientlycorrelated by fluorescent levels in living ST fish. Some sites ofinfections were uncovered in the ST fish were initially missed withclassical histology, such as in the peritoneal lining, pancreas, fat andswim bladders. These unexpected sites of infections may be the longsought-after hide-outs for latent bacteria. For human TB, all but asmall subpopulation of the infectious load is killed by conventionalantibiotic treatments. These hidden niches are thought to offermicro-environments where the latent “persisters” are all but refractoryto killing by antibiotics.

For eradication this small but persistent subpopulation requires a sixmonth treatment of a cocktail of three antibiotics. Failure to eradicatethe persisters can lead to a relapse of TB, because bacteria eventuallycirculate from these infectious reservoirs and then reinoculate andthose tissues where bacteria were successfully treated by antibiotics.Chronically infected ST fish were treated with two established anti-TBdrugs, isoniazid and rifampin. These treatments provide insights intothe anatomical locations where persistent M. marinum subpopulationsreside. The regions of ST fish where the susceptible fluorescentbacteria are effectively killed by antibiotic treatment (i.e.,“lights-out”) were carefully monitored.

Tissues are being identified where fluorescent bacteria persist(“lights-on”) despite antibiotic treatments. In situ imaging of these“lights-on” subpopulations can document the locations of thesepersistent reservoirs of bacteria. Directly monitoring thesubpopulations residing in each infected tissue can allow forsimultaneously evaluation of the relative efficacy of different anti-TBdrugs in all of the infected tissues. Monitoring the response of thelights-on subpopulations to different antibiotics allows for discoveringdrugs that can efficiently target latent persisters.

The techniques described above allow for the investigation of whetherchronic infections by either Brucella or Mycobacterium increase cancerrisk to the host due to increased mutational loads in and aroundinfected tissues. Human epidemiological studies have long correlatedgreater cancer risk for populations with chronic infectious diseases.For example, increased human risk has been best established for stomachcancer and chronic Helicobacter pylori infections also increasedbladder/colon cancer and chronic Shistosomal infections. Increases inlung cancer has also been correlated with long-term TB patients. Theincreased risk of cancer is believed to stem from persistent,self-inflicted “bystander” DNA damage to host tissues from mutagensgenerated by phagocytes, like macrophages as well as other components ofthe immune system.

This connection between chronic inflammation associated with Brucella orMycobacterium infections and increased cancer risk can be assessed bythe techniques described above. Studies have found that chronicMycobacterium marinum infections yielded approximately a five-foldincrease in hepatocarcinoma frequency in medaka. These studies are beingextended in medaka by testing the effects of variables such as, diet,stress and anti-inflammatory drugs that might influence cancer risks ofchronically-infected animals. These studies are also being used toinvestigate the effects of known host tumor suppressors, DNA repairactivities and error-prone DNA polymerases on mycobacterial-inducedcancers. Colonies of fish carrying defined genetic mutations are beingestablished in the following repair functions: p53, REV1 and RIC1. Thesemutant animals will be infected with M. marinum and the effects onhepatocarcinoma will be evaluated.

Example 7 Influences of Polymer Chemistry on Monocytic Uptake ofNanospheres

Polyanhydride copolymer chemistry (polymer structural composition andproperties) affects the uptake and intracellular compartmentalization ofnanospheres by THP-1 human monocytic cells. Polyanhydride nanosphereswere prepared by an anti-solvent nanoprecipitation technique, asdescribed in the examples above. Morphology and particle diameter wereconfirmed via scanning election microscopy and quasi-elastic lightscattering, respectively. The effects of varying polymer chemistry onnanosphere and fluorescently labeled protein uptake by THP-1 cells weremonitored by laser scanning confocal microscopy.

Polyanhydride nanoparticles composed of poly(sebacic anhydride) (SA),and 20:80 and 50:50 copolymers of 1,6-bis-(p-carboxyphenoxy)hexane (CPH)anhydride and SA were fabricated with similar spherical morphology andparticle diameter (200 to 800 nm). Exposure of the nanospheres to THP-1monocytes showed that poly(SA) and 20:80 CPH:SA nanospheres were readilyinternalized whereas 50:50 CPH:SA nanospheres had limited uptake. Thechemistries also differentially enhanced the uptake of a red fluorescentprotein-labeled antigen.

Accordingly, nanosphere and antigen uptake by monocytes can be directlycorrelated to the chemistry of the nano sphere. These resultsdemonstrate the importance of choosing polyanhydride chemistries thatfacilitate enhanced interactions with bacterial infections and/orantigen presenting cells that are necessary in the initiation ofefficacious immune responses.

Bioerodible polymers have been studied as sustainable drug deliveryvehicles for over thirty years (1). Polyesters and polyanhydrides aretwo families of polymers that are strong candidates for biomedicalapplications because of the biocompatibility and bioresorbability oftheir degradation products (2). While polyesters, likepoly(lactic-co-glycolic acid) (PLGA), have been approved by the FDA formany in vivo applications (3), their suitability for use as drug orvaccine delivery vehicles and disease treatment is affected by variousfactors that negatively impact the stability of encapsulated proteins.Research has shown that the bulk-erodible polyester-based deliverysystems display rapid release profiles (4,5), produce low pHmicroenvironments (6-8), and can initiate moisture-induced proteinaggregation (8-10).

In contrast, polyanhydrides are characterized by chemistry-dependentsurface erosion and payload release (11-13), moderate pH microenvironments (8,14,15), and superior protein stabilization capabilities(10,16,17). Polyanhydrides have been used to deliver plasmid DNA (18),proteins (9,13,17), small molecular weight drugs (11,19,20), and vaccineimmunogens (21,22). Alterations of polyanhydride chemistry modulatedegradation rates from weeks to years, which can be exploited to bestfit therapeutic needs (9,11,16). In addition, polyanhydride microspheresused as vaccine delivery vehicles exhibit a chemistry-dependent,immunomodulatory adjuvant effect (22). Kipper et al. showed thatencapsulating tetanus toxoid (IT) into polyanhydride microspheres orco-delivering free IT along with the microspheres enhancedantigen-specific immune responses (22). Furthermore, the relativeincrease of polymer hydrophobicity effectively modulated the immuneresponse from a dominant T_(H)2 (humoral) to a T_(H)0 (balanced)response. Together, these results indicate that polyanhydridemicrospheres are promising vehicles for vaccine delivery.

The polyanhydride chemistries used in the present study includesexamples such as copolymers of sebacic anhydride (SA) and1,6-bis(p-carboxyphenoxy)hexane (CPH) anhydride. With aromatic rings,the CPH unit is more hydrophobic than the aliphatic SA unit. Copolymerscontaining higher compositions of CPH have been shown to degrade slowerthan copolymers containing higher compositions of SA (9).

In the last several decades, the in vivo applications utilizing polymercarriers have transitioned from the use of large, implanted pellets (˜1mm) to microspheres (˜5-20 μm) and, more recently, to nanospheres(˜100-500 nm) (1,11). In comparison to implants, microspheres (ornanospheres) do not require surgical insertion or removal (22), cancarry multiple drugs (20,23), and are phagocytosed by antigen presentingcells (APCs) (24). Inhalation and intranasal delivery can be realizedwith particles that are small enough to pass through the finely porousnetworks of the nasal, tracheal, and pulmonary filtration systems(25,26). In addition, multiple studies have shown that polymericnanoparticles gain ready access into sub-mucosal layers of thenasal-associated and gut-associated lymphoid tissues much moreeffectively than microparticles (27-29). In comparison to microspheres,nanospheres were more readily taken up by APCs (30). Collectively, thesecharacteristics underpin the functional diversity and enhancedcapabilities of polyanhydride nanospheres.

For polyanhydride nanospheres to function as efficacious antimicrobialdrug delivery devices and vaccine adjuvants, they must possess theability to deliver the antimicrobial agent into the microbes, and tostimulate and to deliver antigen to APCs, respectively. In the presentstudy, confocal microscopy was used to monitor both intracellular andextracellular interactions between polyanhydride nanospheres and APCs(31). In addition, confocal microscopy allowed for monitoring theability of polyanhydride nanospheres to deliver antigens orantimicrobial agents via the endocytic pathway by evaluating theco-localization of polyanhydride nanospheres within specificsub-cellular compartments associated with antigen processing andpresentation and pathogenic infection sites. Data demonstrate thatsystematically varying the chemistry of polyanhydride nanospheres (byvarying the SA content in a CPH:SA copolymer) significantly affectsnanosphere uptake by human monocytic cells. In addition, it wasdemonstrated that polymer chemistry significantly influences the uptakeof a model antigen (Ea tagged with red fluorescent protein (RFP),henceforth referred to as Eα-RFP) by human monocytic cells.

Preparation of SA and CPH pre-polymers, copolymers, and nanospheres wascarried out, for example, as described in Example 1.

Culture of THP-1 Human Monocytes and Co-Incubation with Nanospheres.

Tissue culture and subsequent derivation of adherent THP-1 monocytes wasperformed according to published reports (34) with some modification(35). Briefly, THP-1 cells were grown in suspension using RPMI 1640growth medium supplemented with 10% newborn calf serum, 10 mM Glutamax,25 mM HEPES, and 10 μ/mL penicillin-streptomycin antibiotics (completeRPMI). Adherent monocytes were derived from suspension cultures bystimulating cells with 5 nM phorbol-12-myristic-13-acetate (PMA) in 24well tissue culture plates containing 10 mm glass coverslips inside eachwell at a final density of 5×10⁵ cells per well. Following 24 hours PMAincubation, cultures were washed with PBS and incubated in fresh RPMIwithout PMA for 24 hours before nanospheres were added.

Polyanhydride nanospheres (in the form of dry powder) of poly(SA), 20:80CPH:SA, or 50:50 CPH:SA were weighed and added to PBS (pH 7.4) at astock concentration of 10 mg/mL. The nanospheres were briefly sonicatedon ice for a total process time of 1 minute alternating 10 seconds pulseON, 15 seconds Pulse OFF. Nanospheres (100 μg) were added to cellculture medium (0.5 ml/well), briefly mixed by pipetting before cultureswere returned to the incubator (37° C., 5% CO₂). To evaluate phagocyticprocesses, the nanospheres were co-incubated with the THP-1 cells for 30minutes. Cultures were washed and the cells were placed back in theincubator for 2 hours prior to analysis. To evaluate endocyticprocesses, the nanospheres were co-incubated with the THP-1 cells for 6hours. Cultures were washed and the cells were placed back in theincubator for 48 hours prior to analysis.

Fluorescence Microscopy Techniques.

To observe time-dependent interactions of individual monocytes withnanospheres, cell monolayers incubated with nanospheres at indicatedtimes were fixed with 4% para-formaldehyde (PFA) for 10 minutes at roomtemperature, and then washed with PBS. Acidic vesicles and lipid raftsin cell monolayers were labeled by incubating cells for 20 minutes priorto fixation with either Lysotracker at 1/2,000 dilution (DND-99)(acidicvesicles) or Alexa555 conjugated Cholera Toxin β-subunit (CTx) at 1/150dilution (lipid rafts) (Molecular Probes-Invitrogen, Carlsbad, Calif.).Intracellular structures were immunofluorescently stained by incubatingfixed coverslips with primary and secondary antibodies in PBS containingalbumin and 0.1% saponin (BSP) (35). Stained coverslips were washed andmounted on glass slides (Pro-Long w/Dapi; Molecular Probes-Invitrogen).

Epifluorescence and immunofluorescence microscopy was performed usingeither an Olympus IX-61 inverted microscope equipped with blue, green,and red filter sets with a cooled CCD camera or by an inverted Leica NTSlaser scanning confocal microscopy (LSCM). The LSCM was equipped withApoChromatic x63 oil and x100 oil objectives and UV, Argon, Krypton andHeNe laser lines equipped with three photomultiplier detection tubes.Optimal step size for Z-stack image data was determined empirically frompilot studies to be 0.3 μm. Co-localization analysis, relativenanosphere uptake comparisons, and final images were prepared usingImage J v1.36b image analysis software loaded with particle countingalgorithms (36).

Eα-RFP Antigen Preparation and Cellular Internalization by Monocytes.

The IPTG inducible Eα-RFP expression construct (37) was introduced intoEscherichia coli DH5α by heat shock followed by selecting 50 mg/mLampicillin-resistant colonies. Broth cultures of transformed bacteriawere induced by the addition of IPTG to overnight cultures. Crude celllysates prepared using the Novagen Bugbuster extraction reagent(Gibbstown, N.J.) were passed through a Profinity IMAC Ni-charged resin(BioRad; Hercules, Calif.). Imidazole eluted Eα-RFP protein was dialyzedovernight at 4° C. and final preparations were shown to be free ofdetectable LPS contamination by the limulus ameobocyte lysate (LAL)assay (Cambrex; Walkersville, Md., USA). Fluorescence signal intensityof internalized protein was detected using standard epi-fluorescencemicroscopy employing TRITC/rhodamine filter set with 510-560 nmexcitation and 575-645 emission. Image black levels for the RFP proteinwere set using cells not incubated with RFP. Exposure times for RFPdetection were kept constant throughout the experimental groups tofacilitate accurate comparative analysis. Bar graphs of the relativepixel intensity of internalized RFP were calculated from RAW-RFP imagesusing the ImageRplugin/histogram function. These bar graphs reveal therelative pixel intensity of the RFP protein detected in the presence andabsence of nanospheres.

Nanosphere Characterization.

Scanning electron photomicrographs of the FITC-dextran loadednanospheres of varying formulations are presented in FIG. 10. Thephotomicrographs show that the nanoparticles are spherical, and whilethere are some small variations, the nanospheres appear to be relativelyuniform in size and shape. Light scattering size distribution data shownanosphere diameters for all polymers fall between 200 and 800 nm.

Each batch of nanospheres was analyzed by light scattering and particlesize was measured using duplicate samples. For each polymer chemistry,the data from three different lots of nanospheres were analyzed in thismanner and the compiled data are shown in Table I. The standarddeviations were determined for the overall accumulated size distributiondata for each polymer.

TABLE I Particle Size Data Compiled from Light Scattering Measurements(n = 3). Average Particle Polymer Diameter (nm) poly SA 283 ± 45 20:80CPH:SA 348 ± 48 50:50 CPH:SA  397 ± 121 Data reported as mean ± SD.

Analysis shows that there is no statistically significant difference inaverage particle size among the different polymer formulations (p=0.13).This data demonstrates that polyanhydride nanospheres fabricated by thePAN method can be reproducibly prepared with similar morphology andparticle diameters regardless of copolymer chemistry. Having particlesof similar size is important in limiting the variables that areintroduced into in vitro and in vivo experiments, especially whenevaluating a polymer chemistry effect. While not statisticallysignificant, there was a slight trend for a positive correlation betweenparticle size and CPH content.

The thermodynamic and kinetic balance between nucleation and growthdictates the resulting average particle size. The soluble material mustnucleate particles and then more material can either precipitate on thesurface of these already formed particles or new particles can benucleated (38). Copolymers with a higher SA content are less hydrophobicand more non-polar than those with a higher CPH content. Whenprecipitating from a polar solvent into an aliphatic antisolvent bath,copolymers with a higher SA content may more easily nucleate newparticles. If nucleation is favored, it would cause more particles to beformed with a smaller average particle size.

Cellular Interactions of Nanospheres with Human Monocytes.

To determine whether polymer chemistry affects nanoparticleinternalization and intracellular deposition within APCs, adherent humanTHP-1 monocytes were incubated separately with poly(SA), 20:80 CPH:SA,or 50:50 CPH:SA nanospheres. LSCM was utilized to evaluate and comparethe interactions of nanospheres with cells and their eventualintracellular localization.

Internalization.

Nanospheres introduced into cell culture medium did not form largeaggregates and remained uniformly dispersed prior to settling at thebottom of the tissue culture well during co-incubation with the THP-1cells. The nanospheres were then rapidly internalized by THP-1 monocytesvia cellular events consistent with phagocytosis (FIG. 11). Observationssupporting this conclusion include centrifugation-independentinternalization, temperature-dependent internalization, andinternalization in the absence of an overabundance of extracellularparticles.

Confocal photomicrographs in FIG. 11 depict monocytes that haveinternalized nanospheres and values presented in Table II indicate thepercentage of THP-1 cells per field of view that have cell associatednanospheres at 2 or 48 hours post exposure. Cells were imaged at 1000×total magnification and the average number of cells in each Field ofView (FOV) was 25. FOVs were randomly selected and the numbers of THP-1cells with FITC-loaded polyanhydride nanospheres or without nanosphereswere recorded. The percentages and standard deviations of THP-1 cellspositive for nanospheres were calculated from values for ≧5 FOV imagesfor each nanosphere chemistry and time point (cells withFITC-nanospheres/total # cells scored). The total cells scoredpositively for clear association with FITC nanospheres were combinedfrom data collected over three to five independent experiments.

TABLE II Association of Polyanhydride Nanospheres with THP-1 CellsVaries Depending on Polymer Chemistry. Percent monocytes withinternalized nanospheres^(a) Polymer 2 h (phagocytosis) 48 h(phagocytosis and endocytosis) Poly(SA) 87.9% ± 17.1% 96.3% ± 11.7%20:80 CPH:SA 27.1% ± 14.8% 91.2% ± 22.2% 50:50 CPH:SA  8.1% ± 10.6%53.1% ± 28.3  ^(a)Average percent nanospheres positive monocytescalculated per x 100 field of view image.

The data in Table II indicate that in the experiments designed toevaluate phagocytosis where the exposure to nanospheres was 30 minutes,the least hydrophobic polymers (i.e., poly(SA)) were more rapidlyinternalized than the more hydrophobic (i.e., CPH-containing) polymers(FIG. 11). In contrast, the 48 hours co-localization experiments employa longer exposure time of nanospheres with cells lasting 6 hours. Inthese experiments, where endocytosis plus the initial phagocytosis wouldcontribute to total nanosphere uptake, it was observed that ˜96% of theTHP-1 cells contained poly(SA) nanospheres, while the uptake of 20:80CPHSA and 50:50 CPH:SA was ˜91% and ˜53%, respectively. These resultsindicate that the polymer chemistry of the polyanhydride nanospheresaffects the uptake efficiency of these nanospheres by monocytes.

Unlike CPH-containing nanospheres, poly(SA) nanospheres were moreefficiently internalized by phagocytic processes (30 minutes exposure tocells) and did not require the extended time (6 hours) associated withendocytic processes. The more hydrophobic nanospheres (i.e., CPH-rich)were not internalized by phagocytic pathways (˜8%). However, with time,all the formulations were internalized; but, 50:50 CPH:SA nanosphereswere internalized to a lesser extent (˜53%, Table II). Overall, monocyteuptake of polyanhydride nanospheres correlated with decreasinghydrophobicity (poly(SA)>20:80 CPHSA>50:50 CPHSA).

The degree of hydrophobicity is a significant factor influencingnanosphere uptake. The hydrophobic nature of these particles canfacilitate their interaction with hydrophobic lipid-rich micro-domainsin the cell membrane, including lipid rafts. Lipid rafts contain manymembrane-bound cofactors that comprise receptor complexes, such asreceptors for complement, antibodies, and serum and extracellular matrixproteins (39-41). In contrast with phagocytosis, increasing polymerhydrophobicity can facilitate closer nanosphere-to-cell interactions andincrease the probability of internalization through constitutiveendocytic or macropinocytotic pathways. These hydrophobic interactionsfacilitate nanosphere internalization by direct association with surfacereceptors or through direct interactions with the plasma membrane.

Pattern recognition receptors (PRRs) are another key receptor type foundin lipid rafts of APCs. PRRs recognize pathogen-associated molecularpatterns (PAMPs), which are repetitive patterns of molecular structurefound in both microorganisms and the mammalian host. Examples of PAMPsinclude lipopolysaccharide and flagellin from bacteria as well ashyaluronan and uric acid from mammals. All of these PAMPs signal“danger” to the host, be it in the context of infection or cellulardamage. Hydrophobic characteristics have been ascribed to many PAMPs andare thought to be partly responsible for their immunostimulatoryproperties (42). In the context of the polyanhydride co-polymers,surface patterns of intervening hydrophobic moieties (e.g., CPH and SA)may mimic PAMPs, facilitate interactions with PRRs present on thesurface of APCs and subsequently enhance the ability of APCs to activateT cells (43, 44). Internalization and co-localization of antigen-loadednanospheres within the endocytic pathway may, in part, explain theadjuvanticity of polyanhydride nanospheres (22).

Intracellular Localization.

In general, intracellular degradation and processing of exogenouslypresented antigen occurs when lysosomes fuse with late endosomescontaining antigen. In contrast, endogenous antigen is processed withinthe cytosol by the proteosome (45). As a result, antigen fate (i.e., MHCI vs MHC II presentation) is largely decided by intracellular location.Given the variable surface chemistry presented by the differentpolyanhydrides, the intracellular distribution of nanospheres 48 hoursafter uptake was analyzed. The majority of particles were found to beintact and located within membrane bound vesicles that werecharacterized as acidic and CTx⁺ (FIG. 12). In color versions of thesephotomicrographs, the FITC-dextran containing nanospheres appear green,the acidic vesicles are red (Lysotracker), and co-localized nanosphereswithin acidic vesicles appear yellow. The data and images clearlyindicate that the polyanhydride chemistries studied resulted inlocalization of the nanospheres into the acidic phagolysosomalcompartments of the cells.

The majority of these particles were rapidly targeted to the endosomalpathway, and localized within vesicles exhibiting stainingcharacteristics and morphology consistent with MHC class II loadingcompartments (46). At 48 hours, ˜10% of the poly(SA) and 20:80 CPH:SAnanospheres did not appear to be located within acidic or lipid raftcontaining vesicles (FIG. 11). A lack of localization within either ofthese major intracellular compartments is consistent with nanospheresthat are free within the cellular cytosol. Release of antigen fromnanospheres located within the cellular cytosol would be processed anddirected to the MHC class I presentation pathway (45). However, the datapresented in FIGS. 11 and 12 indicate various amounts of nano spherescan reach the cytosol.

Antigen Internalization.

As previously discussed, polyanhydride nanospheres serve as antigendelivery platforms to APCs. Nanosphere-encapsulated immunogens can bereleased intracellularly following internalization and slow polymerdegradation (8). However, some nanospheres may release antigen prior touptake, providing a source of soluble antigen delivered to APCs viaendocytosis. To evaluate the ability of nanospheres to stimulate solubleantigen internalization by APCs, the THP-1 cells were co-incubated withblank nanospheres (poly (SA), 20:80 CPH:SA, or 50:50 CPH:SA) and solubleEα-RFP (37), fixed, and visualized by epifluorescence microscopy.Representative photomicrographs and bar graphs summarizing cellassociated RFP data are provided in FIG. 9.

Comparisons among the three chemistries reveal that after 2 hours ofco-incubation, all three chemistries dramatically increased the amountof soluble antigen internalized by monocytes. A potential mechanism forthe increase in uptake stimulated by the nanospheres is that the proteinitself is able to adsorb on the surface of nanospheres that are thensubsequently internalized by the APC. However, preliminary experimentsfailed to detect soluble RFP adsorbing onto FITC-labeled nanospheres andculture conditions include ample amounts of serum proteins present inthe 10% fetal bovine serum supplemented medium. Moreover, the dramaticincrease in the uptake of soluble RFP was also detected for 50:50 CPH:SAeven though these particles serve as poor targets for uptake themselves(Table II, FIGS. 11 and 12). This data demonstrates that the polymerchemistry of the polyanhydride nanospheres influences the ability ofAPCs to internalize soluble antigen.

Accordingly, the unique cellular interactions elicited by polyanhydridenanospheres are a function of the particles' distinct physical andchemical properties that modulate the persistence and intracellulardistribution of antigen. Polyanhydride nanospheres were internalized anddistributed within human monocytes in a chemistry-dependent manner.Chemical structure of the polymers also influences the ability ofnanospheres to enhance monocytic uptake of soluble antigen. Together,this data highlights the importance of chemistry in designingpolyanhydride nanospheres as vaccine or drug delivery vehicles intendedfor specific applications and/or targeting desired intracellularlocations. Thus, the drug encapsulated nanoparticles can be effectivelyused to deliver active agents to target cells to inhibit microbe growthand to deliver vaccines.

Example 7 Citations

-   1. I. Preis, and R. S. Langer. A single-step immunization by    sustained antigen release. J. Immunol. Methods. 28(1-2):193-197    (1979).-   2. J. H. Wilson-Welder et al. Vaccine adjuvants: Current challenges    and future approaches. J. Pharm. Sci. 2008.-   3. S. P. Schwendeman. Recent advances in the stabilization of    proteins encapsulated in injectable PLGA delivery systems. Crit.    Rev. Ther. Drug Carr. Syst. 19:73-98 (2002).-   4. A. Gopferich. Polymer bulk erosion. Macromolecules. 30:2598-2604    (1997).-   5. Y. Wang et al. Controlled release of ethacrynic acid from poly    (lactide-co-glycolide) films for glaucoma treatment. Biomaterials.    25(18):4279-4285 (2004).-   6. K. Fu et al. Visual evidence of acidic environment within    degrading poly(lactic-co-glycolic acid) (PLGA) microspheres. Pharm.    Res. 17(1):100-106 (2000).-   7. A. G. Ding, and S. P. Schwendeman. Acidic microclimate pH    distribution in PLGA microspheres monitored by confocal laser    scanning microscopy. Pharm. Res. 25(9):2041-2052 (2008).-   8. A. S. Determan et al. Protein stability in the presence of    polymer degradation products: consequences for controlled release    formulations. Biomaterials. 27(17):3312-3320 (2006).-   9. A. S. Determan et al. Encapsulation, stabilization, and release    of BSA-FITC from polyanhydride microspheres. J. Control. Release.    100(1):97-109 (2004).-   10. G. Zhu, S. R. Mallery, and S. P. Schwendeman. Stabilization of    proteins encapsulated in injectable poly(lactic-co-glycolic acid).    Nat. Biotechnol. 18:52-57 (2000).-   11. M. J. Kipper et al. Design of an injectable system based on    bioerodible polyanhydride microspheres for sustained drug delivery.    Biomaterials. 23(22):4405-4412 (2002).-   12. E. Shen et al. Mechanistic relationships between polymer    microstructure and drug release kinetics in bioerodible    polyanhydrides. J. Control. Release. 82(1):115-125 (2002).-   13. E. Ron et al. Controlled release of polypeptides from    polyanhydlides. Proc. Natl. Acad. Sci. USA. 90(9):4176-4180 (1993).-   14. L. Shieh et al. Erosion of a new family of biodegradable    polyanhydrides. J. Biomed. Materi. Res. 28(12):1465-1475 (1994).-   15. J. P. Jain et al. Role of polyanhydrides as localized drug    carriers. J. Control. Release. 103(3):541-563 (2005).-   16. A. S. Determan et al. The role of microsphere fabrication    methods on the stability and release kinetics of ovalbumin    encapsulated in polyanhydride microspheres. J. Microencapsul.    23(8):832-843 (2006).-   17. Y. Tabata, S. Gutta, and R. Langer. Controlled delivery systems    for proteins using polyanhydride microspheres. Pharm. Res.    10(4):487-496 (1993).-   18. B. A. Pfeifer et al. Poly(ester-anhydride):poly(beta-amino    ester) microspheres and nanospheres: DNA encapsulation and cellular    transfection. Int. J. Pharm. 304(1-2):210-219 (2005).-   19. N. B. Shelke, and T. M. Aminabhavi. Synthesis and    characterization of novel poly(sebacic anhydride-co-Pluronic    F68/F127) biopolymeric microspheres for the con trolled release of    nifedipine. Int. J. Pharm. 345(1-2):51-58 (2007).-   20. W. Hsu et al. Local delivery of interleukin-2 and adriamycin is    synergistic in the treatment of experimental malignant glioma. J.    Neurooncol. 74(2):135-140 (2005).-   21. J. Hanes, M. Chiba, and R. Langer. Degradation of porous poly    (anhydride-co-imide) microspheres and implications for controlled    macromolecule delivery. Biomaterials. 19(1-3):163-172 (1998).-   22. M. J. Kipper et al. Single dose vaccine based on biodegradable    polyanhydride microspheres can modulate immune response    mechanism. J. Biomed. Materi. Res. Part A. 76(4):798-810 (2006).-   23. C. Berkland et al. Microsphere size, precipitation kinetics and    drug distribution control drug release from biodegradable    polyanhydride microspheres. J. Control. Release. 94(1):129-141    (2004).-   24. F. X. Lacasse et al. Influence of surface properties at    biodegradable microsphere surfaces: effects on plasma protein    adsorption and phagocytosis. Pharm. Res. 15(2):312-317 (1998).-   25. J. A. Schwab, and M. Zenkel. Filtration of particulates in the    human nose. Laryngoscope. 108(1):120-124 (1998).-   26. P. A. Jaques, and C. S. Kim. Measurement of total lung    deposition of inhaled ultrafine particles in healthy men and women.    Inhal. Toxicol. 12(8):715-731 (2000).-   27. M. P. Desai et al. Gastrointestinal uptake of biodegradable    microparticles: effect of particle size. Pharm. Res.    13(12):1838-1845 (1996).-   28. T. Jung et al. Tetanus toxoid loaded nanoparticles from    sulfobutylated poly(vinyl alcohol)-gradt-poly(lactide-co-glycolide):    evaluation of antibody response after oral and nasal application in    mice. Pharm. Res. 18(3):352-360 (2001).-   29. L. Illum. Nanoparticulate systems for nasal delivery of drugs: a    real improvement over simple systems? J. Pharm. Sci. 96(3):473-483    (2007).-   30. M. P. Desai et al. The mechanism of uptake of biodegradable    microparticles in Caco-2 cells is size dependent. Pharm. Res.    14(11):1568-1573 (1997).-   31. J. E. Fuller et al. Intracellular delivery of core-shell    fluorescent silica nanoparticles. Biomaterials. 29(10):1526-1532    (2008).-   32. A. Conix. Poly[1,3-bis(p-carboxyphenoxy)propane anhydride).    Macromolecular Synthesis. 2:95-98 (1966).-   33. E. Mathiowitz et al. Biologically erodible microspheres as    potential oral drug delivery systems. Nature. 386(6623):410-414    (1997).-   34. R. W. Stokes, and D. Doxsee. The receptor-mediated uptake,    survival, replication, and drug sensitivity of Mycobacterium    tuberculosis within the macrophage-like cell line THP-1: a    comparison with human monocyte-derived macrophages. Cell. Immunol.    197(1):1-9 (1999).-   35. B. H. Bellaire, R. M. Roop II, and J. A. Cardelli. Opsonized    virulent Brucella abortus replicates within nonacidic, endoplasmic    reticulum-negative, LAMP-1-positive phagosomes in human monocytes.    Infecation and Immunity. 73(6):3702-3713 (2005).-   36. ImageJ. Image Processing and Analysis in Java [cited 2008 Aug.    3); Available from: http://rsb.info.nih.gov/ij/.-   37. A. A. Itano et al. Distinct dendritic cell populations    sequentially present antigen to CD4 T cells and stimulate different    aspects of cell-mediated immunity. Immunity. 19(1):47-57 (2003).-   38. E. Mathiowitz, et al. Process for preparing microparticles    through phase inversion phenomena. 2003: United States of America.-   39. P. Lajoie, and I. R. Nabi. Regulation of raft-dependent    endocytosis. J. Cell. Mol. Med. 11(4):644-653 (2007).-   40. N. Gupta, and A. L. DeFranco. Visualizing lipid raft dynamics    and early signaling events during antigen receptor-mediated    Blymphocyte activation. Mol. Biol. Cell. 14(2):432-444 (2003).-   41. Z. Wolf et al. Monocyte cholesterol homeostasis correlates with    the presence of detergent resistant membrane microdomains. Cytometry    Part A. 71(7):486-494 (2007).-   42. S. Y. Seong, and P. Matzinger. Hydrophobicity: an ancient    damage-associated molecular pattern that initiates innate immune    responses. Nat. Rev. Immunol. 4(6):469-478 (2004).-   43. M. G. Netea et al. From the Th1/Th2 paradigm towards a Toll-like    receptor/T-helper bias. Antimicrob. Agents Chemother.    49(10):3991-3996 (2005).-   44. P. Elamanchili et al. “Pathogen-mimicking” nanoparticles for    vaccine delivery to dendritic cells. J. Immunother. 30(4):378-395    (2007).-   45. A. L. Goldberg et al. The importance of the proteasome and    subsequent proteolytic steps in the generation of antigenic    peptides. Mol. Immunol. 39(3-4):147-164 (2002).-   46. E. M. Hiltbold, and P. A. Roche. Trafficking of MHC class II    molecules in the late secretory pathway. Curr. Opin. Immunol.    14(1):30-35 (2002).

Other citations that provide useful information include:

-   1. Lecaroz et al. Poly(D,L-lactide-coglycolide) particles containing    gentamicin: pharmacokinetics and pharmacodynamics in Brucella    melitensis-infected mice. Antimicrob. Agents Chemother. 2007, 51:    1185-1190.-   2. Bellaire et al. The siderophore 2,3-dihydroxybenzoic acid is not    required for virulence of Brucella abortus in BALB/c mice. Infect.    Immun. 1999, 67: 2615-2618.-   3. Baldwin and Parent. Fundamentals of host immune response against    Brucella abortus: what the mouse model has revealed about control of    infection. Vet. Microbiol. 2002, 90:367-382.-   4. Bellaire B H, Roop R M, 2nd, Cardelli J A. Opsonized virulent    Brucella abortus replicates within nonacidic, endoplasmic    reticulum-negative, LAMP-1-positive phagosomes in human monocytes.    Infect. Immun. 2005, 73: 3702-3713.-   5. Rittig et al. Intracellular survival of Brucella spp. in human    monocytes involves conventional uptake but special phagosomes.    Infect Immun 2001, 69: 3995-4006.

Example 8 Polyanhydride Nanospheres Provide Improved AntibioticTreatments

The polyanhydride nanospheres described herein demonstrated improvedantibiotic activity of doxycycline and other antibiotics to killintracellular, virulent Brucella abortus 2308 in human and mousemonocytes. The polyanhydride nanospheres also demonstrated sustainedrelease with retained antimicrobial activity using disk diffusion assayagainst virulent Brucella canis. Increased antibiotic activity onbacteria in culture alone (not associated with infection) was observed.The increase was approximately 30× greater than soluble doxycyclinealone. Thus, encapsulation of antibiotics could effectively treat avariety of bacteria, including Antibiotic Resistant MethacillinResistant Staphylococcus aureus (MRSA), Extremely Drug ResistantMycobacterium tuberculosis (XTBR), Gram positive bacteria such asStreptococcus and Bacillus, protozoans such as Leishmania, and envelopedviruses such as influenza and HIV. The particles were confirmed to bestable within dendritic cells and monocytes. Data indicated that theparticles slowly degrade over more that 5 days with continued release ofthe cargo antimicrobial agents.

FIG. 13 illustrates the enhanced killing of intracellular B. abortus byanalysis of a viability experiment at 72 hours post-inoculation, where20:80 CPH:SA and 20:80 CPTEG:CPH polyanhydride particles withdoxycycline cargo were significantly more effective at killing B.abortus than doxycycline solubilized in a PBS solution. The killing ofintracellular Brucella abortus 2308 within human monocytes was enhancedthrough antibiotic encapsulation. Human monocytes cultures were infectedwith virulent B. abortus 2308 to establish a productive intracellularinfection. At 24 hours, infected cultures were supplemented with 10μg/mL of doxycycline in either soluble form (PBS solution) orencapsulated in PA polymers (vertical arrow in FIG. 13). Following anadditional 48 hours of incubation (t=72 hours, total infection),non-treated and drug treated cultures were washed, and lysed to releaseintracellular bacteria, which were subsequently diluted serially andplated on solid agar medium.

The antimicrobial agent (e.g., doxycycline) was either solubilized in aPBS solution or used by preparing a slurry of PA particles in a PBSsolution. The concentration (μg/mL) an antimicrobial agent wascalculated by preparing PA particles with 5 wt. % antibiotic in theparticles. Then a proportional amount of PA particles were added to aPBS buffer (e.g., 20× the mass of particle compared to the mass ofantimicrobial agent directly dissolved in the PBS buffer).

FIG. 14 shows the continued antibiotic release by serial disc transferof polyanhydride particles containing doxycycline. Antibiotic releasefrom cellulose filter disks receiving soluble doxycycline or equivalentamounts of PA nanoparticle encapsulated doxycycline was analyzed. Liquidsolutions of either soluble doxycycline or PA-encapsulated doxycyclinewere place onto filter paper to conduct standard zone of inhibitionmeasurements on agar Brucella canis spread plates. After 24 hours ofincubation, zones of inhibition were measured and the filter disks wereaseptically removed and placed onto fresh B. canis spread plates tomeasure zones of inhibition following an additional 24 hours ofincubation. Only PA nanoparticle encapsulated doxycycline filtersretained any residual antibiotic activity following serial transfer.This experiment demonstrates the continued antibiotic release fromdegrading PA nanoparticles.

These data demonstrate steady and delayed release of antibiotic and thatreleased antibiotic retains full antimicrobial activity andeffectiveness against a human and animal pathogen Brucella canis. Thedisc treated with PA nanoparticles continued to provide zones ofinhibition on fresh B. canis spread plates with full activity after 48hours and additional activity at 72 hours. The antimicrobial activity ofthe particles can persist for several days, although the activity beginsto decrease after 72 hours, whereas dissolved antibiotic alone displayedno activity on the disk after one plate treatment.

Particle internalization and stability at 48 hours post internalizationby Murine Bone Marrow Derived Monoctyes was also analyzed. Confocalimages of the intracellular localization of PA-nanospheres were capturedby LSCM and processed using ImageJ. Analysis of the images provided thedate shown in Table 8-1.

TABLE 8-1 Internalization of Polyanhydride Nanoparticles. Percentmonocytes with internalized nanoparticles Polymer 2 h (phagocytosis) 48h (phagocytosis and endocytosis) Poly(SA) 88% ± 17% 96% ± 11% 20:80CPH:SA 27% ± 15% 91% ± 22% 50:50 CPH:SA  8% ± 11% 53% ± 28%

The polyanhydride particles show significant sustained releaseproperties and are more effective at killing microbes for extendedperiods of time with a fraction of an equivalent does of a solubilizedantibiotic.

Example 9 Polyanhydride Nanospheres Provide Enhanced Killing of Microbes

Antibiotics encapsulated in the polyanhydride nanospheres describedherein demonstrate significantly effective killing of Mycobacteriumavium paratuberculosis compared to solubilized antibiotics alone.Examples of the antibiotics tested include amikacin and rifampicin.Other active agents, such as those described herein, can be encapsulatedin the polyanhydride nanospheres to kill microbes in an extracelluarfashion, either in vivo or in vitro, such as in broth and infectedmacrophage cultures. FIGS. 15-21 and Table 9-1 illustrate data obtainedfrom experiments demonstrating the effectiveness of these compositions.

FIG. 15 illustrates viability of Mycobacterium avium subsp.paratuberculosis (MAP) at 24 hours and 48 hours after treatment with PBSsolubilized amikacin or polyanhydride particles containing 10 μg/mLamikacin. The experiments were carried out with amikacin (16 μg/mL),followed by reduction by 1 log. The antibiotic resistance was measuredat days 4 and 7. See Pelligrin (J. Antimicrobial Chemotherapy, 2006) forassessing antimicrobial activity of amikacin on MAP in broth culture.

In the assay used to provide the results illustrated in FIG. 15, thesoluble 10 μg/mL (lower dose) was measured by % live MAP. Directcomparison of standard soluble amikacin added to MAP cultures showedminimal antimicrobial activity. In contrast, amikacin loaded into 20:80CPH:SA nanospheres killed >50% of MAP by 48 hours. Also, amikacin loadedinto 20:80 CPTEG:CPH exhibited >20% killing of MAP. This interactionwith the bacteria was anhydride polymer chemistry dependent as evidentby no antimicrobial activity being detected in cultures treated withamikacin loaded 50:50 CPTEG:CPH nanoparticles.

FIGS. 16 and 17 show data from the results of experiments carried outwith the following treatment groups:

-   -   5% amikaycin (Sigma);    -   #1 20:80 CPH:SA (5% amikaycin);    -   #2 20:80 CPTEG:CPH (5% amikaycin); and    -   #3 50:50 CPTEG:CPH (5% amikaycin).        The data is summarized in Table 9-1.

TABLE 9-1 Broth Time Sample 1.25 μg/mL 10 μg/mL 24 hrs untreated 94.23%93.01% Soluble 93.79% 90.08% #1 20:80 CPH:SA 82.14% 59.74% #2 20:80CPTEG:CPH 86.91% 78.95% #3 50:50 CPTEG:CPH 92.80% 90.34% 48 hrsuntreated 88.91% 94.44% Soluble 88.42% 84.75% #1 20:80 CPH:SA 81.68%43.72% #2 20:80 CPTEG:CPH 83.40% 69.16% #3 50:50 CPTEG:CPH 85.93% 82.44%

FIG. 16 illustrates viability of MAP in broth at 24 hours and 48 hoursafter treatment with PBS solubilized amikacin or polyanhydride particlescontaining 1.25 μg/mL amikacin. FIG. 17 illustrates viability of MAP inbroth at 24 hours and 48 hours after treatment with PBS solubilizedamikacin or polyanhydride particles containing 10 μg/mL amikacin.

Determinations of intracellular bacterial viability and extracellularbacterial killing for this Example were determined as schematicallyillustrated in FIG. 18. Metabolically active MAP are FITC positive.Fluorescence intensity for individual bacteria is recorded by flow andgated against cultures with lysed cells without bacteria. Forward andside scatter was consistent with intact MAP. The % live MAP was measuredfor each sample and two biological replicates were carried out for eachsample.

FIG. 19 illustrates percent viability of intracellular MAP in U937 humanpro-monocytes at 24 hours (left bars) and 48 hours (right bars) aftertreatment with PBS solubilized amikacin or polyanhydride particlescontaining 10 μg/mL amikacin. The 20:80 CPTEG:CPH formulation used inthese experiments had significantly higher killing of intracellular MAPthan soluble amikacin.

FIG. 20 illustrates percent cell viability of intracellular MAP inRAW264 at 24 hours and 48 hours after treatment with PBS solubilizedamikacin or polyanhydride particles containing 10 μg/mL amikacin. Thus,50:50 CPTEG:CPH particles containing an active agent is surprisinglyeffected at killing extracellular microbes.

FIG. 21 illustrates percent viability of intracellular MAP in J774A at24 hours and 48 hours after treatment with PBS solubilized amikacin orpolyanhydride particles containing 10 μg/mL amikacin. Encapsulation ofthe amikacin antibiotic into 20:80 CPTEG:CPH nanoparticle formulationenabled significantly higher antimicrobial activity than the equivalentdose of soluble antibiotic. Moreover, given that antibiotic release isdelayed, this higher degree of killing occurs with an effectively lowerconcentration of available (released) antibiotic. Exact antibioticconcentration released can be quantified by HPLC.

Accordingly, Applicants have demonstrated that particle encapsulationusing polyanhydride copolymers enables higher antimicrobial activityagainst microbes such as Escherichia coli, Yersinia, Brucella andMycobacterium. Encapsulated antibiotics were released from erodingnanoparticles in a controlled manner, evidenced by continualantimicrobial activity using filter disk passage experiments. Optimalcopolymer formulations vary depending on the particular microbe targetedand whether the treatment is to be given in vitro or in vivo. Severalantibiotics have been successfully encapsulated using variable copolymerformulations, and it is believed that virtually any sort of cargo can beencapsulated in the polyanhydride particles, including proteins andbacterial lysates.

Example 10 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a PANS composition described herein (hereinafterreferred to as ‘Composition X’):

(i) Tablet 1 mg/tablet ‘Composition X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0 (ii) Tablet 2 mg/tablet ‘Composition X’ 20.0Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate15.0 Magnesium stearate 5.0 500.0 (iii) Capsule mg/capsule ‘CompositionX’ 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelatinizedstarch 120.0 Magnesium stearate 3.0 600.0 (iv) Injection 1 (1 mg/mL)mg/mL ‘Composition X’ 1.0 Dibasic sodium phosphate 12.0 Monobasic sodiumphosphate 0.7 Sodium chloride 4.5 1.0N Sodium hydroxide solution q.s.(pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (v)Injection 2 (10 mg/mL) mg/mL ‘Composition X’ 10.0 Monobasic sodiumphosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.001N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL (vi) Aerosol mg/can ‘Composition X’ 20 Oleic acid10 Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Composition X’. Aerosol formulation (vi) may be usedin conjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, these embodiments and examplesare only illustrative and do not limit the scope of the invention.Changes and modifications can be made in accordance with ordinary skillin the art without departing from the invention in its broader aspectsas defined in the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A polyanhydride nanoparticle comprising:polyanhydride polymers in the form of a nanoparticle and anantimicrobial agent located in the interior of the nanoparticle, whereinthe nanoparticle is substantially spherical in shape and has an averagediameter of about 100 nm to about 900 nm; wherein the polyanhydridepolymers comprise anhydride copolymers of sebacic anhydride (SA) and1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride units; and wherein theratio of sebacic anhydride (SA) to 1,6-bis-(p-carboxyphenoxy)hexane(CPH) anhydride in the nanoparticle is about 80:20.
 2. The polyanhydridenanoparticle of claim 1 wherein the nanoparticle has a diameter of about200 nm to about 800 nm.
 3. The polyanhydride nanoparticle of claim 1wherein the nanoparticle has a diameter of about 300 nm to about 400 nm.4. The polyanhydride nanoparticle of claim 1 wherein the antimicrobialagent comprises amikacin, bacillomycin, cephalexin, cephalosporin,ciprofloxacin, doxycycline, erythromycin, ethambutol, gentamicin, aheavy metal, isoniazid, penicillin, rifampin, spectinomycin,streptomycin, sulfa, tetracycline, trimethoprim-sulfamethoxazole,vancomycin, or a combination thereof.
 5. The polyanhydride nanoparticleof claim 1 wherein the antimicrobial agent is doxycycline.
 6. Thepolyanhydride nanoparticle of claim 4 wherein the nanoparticle comprisestwo or more different antimicrobial agents.
 7. The polyanhydridenanoparticle of claim 1 wherein the polyanhydride nanoparticleencapsulates an average of about 1 μg/mL to about 12 μg/mL of theantimicrobial agent.
 8. A polyanhydride nanoparticle comprising:polyanhydride polymers in the form of a nanoparticle, and anantimicrobial agent located in the interior of the nanoparticle, whereinthe nanoparticle is substantially spherical in shape, and has an averagediameter of about 200 nm to about 800 nm; wherein the polyanhydridepolymers comprise anhydride copolymers of sebacic anhydride (SA) and1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride units; and the ratio ofsebacic anhydride (SA) to 1,6-bis-(p-carboxyphenoxy)hexane (CPH)anhydride in the nanoparticle is about 80:20; and wherein theantimicrobial agent is amikacin, bacillomycin, cephalexin,cephalosporin, ciprofloxacin, doxycycline, erythromycin, ethambutol,gentamicin, a heavy metal, isoniazid, penicillin, rifampin,spectinomycin, streptomycin, sulfa, tetracycline,trimethoprim-sulfamethoxazole, or vancomycin.
 9. The polyanhydridenanoparticle of claim 8 wherein the nanoparticle has a diameter of about300 nm to about 400 nm.
 10. A method to kill microbes or inhibit thegrowth of microbes comprising: contacting microbes with an effectiveantimicrobial amount of a composition comprising polyanhydridenanoparticles of claim 1; wherein the nanoparticles degrade by surfaceerosion in the presence of the microbes over a period of time to releasethe antimicrobial agents from the interior of the nanoparticles, therebykilling the microbes or inhibiting the growth of the microbes.
 11. Amethod to treat a microbial infection in an animal comprising:administering to an animal in need of such treatment an effectiveantimicrobial amount of a composition comprising polyanhydridenanoparticles of claim 1; wherein the nanoparticles accumulate ininfected monocytes, dendritic cells, or both, and the nanoparticlesdegrade by surface erosion over a period of time to release theantimicrobial agents so as to contact and kill microbes or inhibit thegrowth of microbes causing the infection, thereby treating the microbialinfection.
 12. The method of claim 11 wherein the microbial infection isan infection that causes a chronic disease.
 13. The method of claim 11wherein the microbial infection is a bacterial infection.
 14. The methodof claim 13 wherein the bacterial infection is caused by a bacteriumselected from the group consisting of Bordetella, Borrelia, Brucella,Burkholderia, Chlamydia, Erhlichia, Francisella, Mycobacterium,Rickettsia, Salmonella, or Yersinia.
 15. The method of claim 11 whereinthe microbial infection causes a disease selected from the groupconsisting of Bacterial meningitis, Brucellosis, Erhlichiosis, Glanders,Johne's, mastitis, Legionella, Lyme disease, Mycobacteria diseasecomplex, Mycoplasmosis, Q-fever, Salmonellosis, Shigellosis, orTuberculosis.
 16. The method of claim 11 wherein the antimicrobial agentcomprises amikacin, bacillomycin, cephalexin, cephalosporin,ciprofloxacin, doxycycline, erythromycin, ethambutol, gentamicin, aheavy metal, isoniazid, penicillin, rifampin, spectinomycin,streptomycin, sulfa, tetracycline, trimethoprim-sulfamethoxazole,vancomycin, or a combination thereof.
 17. The method of claim 16 whereinthe polyanhydride nanoparticles further comprise two or more differentantimicrobial agents.